METHOD OF DIAGNOSING POOR SURVIVAL PROGNOSIS COLON CANCER USING miR-181b

ABSTRACT

The present invention provides novel methods and compositions for the diagnosis and treatment of colon cancers. In particular, the present invention provides diagnostics and prognostics for colon (including colon adenocarcinoma) cancer patients, wherein the methods related to measuring miR levels can predict poor survival. The invention also provides methods of identifying inhibitors of tumorigenesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of Ser. No. 12/373,358having a 37 CFR §1.371 filing date of Feb. 11, 2009, which claimspriority to PCT/US2007/015892 filed Jul. 12, 2007, which the benefit ofU.S. Provisional Application Ser. No. 60/807,304 filed Jul. 13, 2006 andSer. No. 60/932,736 filed Jun. 1, 2007, the disclosures of which areincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.PO1-CA76259 and No. PO1-CA81534 awarded by the National CancerInstitute. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Colon adenocarcinoma is a major cause of cancer mortality worldwide.Colorectal cancer is the third most common and second leading cause ofcancer death in the United States. Sporadic colon adenocarcinomasinitiate as adenomas and evolve through a progression of molecular,cellular and histologic changes. While five-year mortality rates havemodestly declined over the last three decades, there is still a need toidentify new prognostic biomarkers and therapeutic targets for thisdisease. Currently, chemotherapy has significant therapeutic value butsurgery is the only curative form of treatment.

Ideal therapeutic targets should be causally associated with disease andamenable to designing therapeutic interventions; whereas idealbiomarkers should be easy to measure and have strong associations withclinical outcomes. MicroRNAs could match both criteria.

MicroRNAs are 18-25 nucleotide, non-coding RNA molecules that regulatethe translation of many genes. Since their discovery, they have beenfound to regulate a variety of cellular processes including apoptosis,differentiation and cell proliferation. MicroRNAs may also have a causalrole in carcinogenesis. MicroRNA expression levels are altered in mosttumor types, including colon tumors. The microRNAs miR-15 and miR-16aare deleted or downregulated in the majority of chronic lymphocyticleukemias. Experimental manipulation of specific microRNAs modulatestumor development in mouse model systems. The prognostic potential ofmicroRNAs has also been demonstrated for chronic lymphocytic leukemia,lung cancer⁸ and neuroblastomas. Aberrant microRNAs expression may becausal to carcinogenesis, inhibiting specific microRNAs may havetherapeutic implications. Modified antisense oligonucleotides can bedesigned to specifically inhibit microRNA function. Antagomirs are onetype of antisense oligonucleotide that has proven effective atinhibiting microRNA function in vivo in mice.

SUMMARY OF THE INVENTION

In one broad aspect, there is provided herein a method of diagnosingwhether a subject has, is at risk for developing, or has a decreasesurvival prognosis for, a colon cancer-related disease. The methodincludes measuring the level of at least one miR gene product in a testsample from the subject, wherein an alteration in the level of the miRgene product in the test sample, relative to the level of acorresponding miR gene product in a control sample, is indicative of thesubject either having, or being at risk for developing, the coloncancer-related disease. In a particular aspect, the at least one miRgene product is selected from the group consisting of miR-20a, miR-21,miR-106a, miR-181b, miR-203 and combinations thereof. In one embodiment,the one miR gene product is miR-21.

In another broad aspect, there is provided herein a method of testingfor at least an initiation of, predisposition to, or decreased survivalprognosis for, a colon cancer-related disease response, which comprises:

(1) determining an expression level of at least one marker in a samplefrom a test subject; the at least one marker including at least one miRgene product selected from the group consisting of miR-20a, miR-21,miR-106a, miR-181b, miR-203 and combinations thereof;

(2) comparing the expression level determined in step (1) with a controlexpression level of the marker in a sample from a healthy subject; and

(3) judging the subject to have a colon cancer-related disease when theresult of the comparison in step (2) indicates that: i) the expressionlevel of the at least marker in the test subject is higher than that inthe control, or ii) the expression level of the at least one marker inthe test subject is lower than that in the control.

The sample can comprise one or more of tissue, blood, plasma, serum,urine, and feces. Also, all method steps can be performed in vitro.

In another broad aspect, there is provided herein a method of diagnosingwhether a subject has, is at risk for developing, or has a decreasesurvival prognosis for, a colon cancer-related disease, comprising:

(1) reverse transcribing RNA from a test sample obtained from thesubject to provide a set of target oligodeoxynucleotides;

(2) hybridizing the target oligodeoxynucleotides to a microarraycomprising miRNA-specific probe oligonucleotides to provide ahybridization profile for the test sample; and

(3) comparing the test sample hybridization profile to a hybridizationprofile generated from a control sample, wherein an alteration in thesignal of at least one miRNA is indicative of the subject either having,being at risk for developing, or having a decreased survival prognosisfor, a colon cancer-related disease.

In a particular aspect, the signal of at least one miRNA, relative tothe signal generated from the control sample, is up- or down-regulated.Also, the microarray can comprise miRNA-specific probe oligonucleotidesfor one or more miRNAs selected from the group consisting of miR-20a,miR-21, miR-106a, miR-181b, miR-203 and combinations thereof.

In another broad aspect, there is provided herein a method of inhibitingtumorigenesis in a subject who has, or is suspected of having, a coloncancer-related disease in which at least one miR gene product selectedfrom the group consisting of miR-20a, miR-21, miR-106a, miR-181b,miR-203 and combinations thereof, is down-regulated or up-regulated inthe cancer cells of the subject, relative to control cells, comprising:

(1) when the at least one miR gene product is down-regulated in thecancer cells, administering to the subject an effective amount of atleast one isolated miR gene product selected from the group consistingof miR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinationsthereof, such that tumorigenesis is inhibited in the subject; or

(2) when the at least one miR gene product is up-regulated in the cancercells, administering to the subject an effective amount of at least onecompound for inhibiting expression of the at least one miR gene productselected from the group consisting of miR-20a, miR-21, miR-106a,miR-181b, miR-203 and combinations thereof, such that tumorigenesis isinhibited in the subject.

In a particular aspect, at least one isolated miR gene product in step(1) and/or in step (2) is miR-21 or an isolated variant orbiologically-active fragment or functional equivalent thereof, or anantibody that binds thereto.

In another broad aspect, there is provided herein a method of inhibitingtumorigenesis in a subject who has a colon cancer, comprising:

(1) determining the amount of at least one miR gene product in cancercells from the subject, relative to control cells; and

(2) altering the amount of miR gene product expressed in the cancercells by:

-   -   (i) administering to the subject an effective amount of at least        one isolated miR gene product selected from the group consisting        of miR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations        thereof, if the amount of the miR gene product expressed in the        cancer cells is less than the amount of the miR gene product        expressed in control cells; or    -   (ii) administering to the subject an effective amount of at        least one compound for inhibiting expression of the at least one        miR gene product, if the amount of the miR gene product        expressed in the cancer cells is greater than the amount of the        miR gene product expressed in control cells, such that        tumorigenesis is inhibited in the subject.

In a particular aspect, the at least one isolated miR gene product instep (i) is miR-21 or an isolated variant or biologically-activefragment thereof. Also, in certain embodiments, the at least one miRgene product in step (ii) is selected from the group consisting ofmiR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof,or an isolated variant or biologically-active fragment thereof.

In another broad aspect, there is provided herein a method ofidentifying an inhibitor of tumorigenesis, comprising providing a testagent to a cell and measuring the level of at least one miR gene productassociated with an altered expression levels in a colon cancer-relateddisease, wherein an increase or decrease in the level of the miR geneproduct in the cell, relative to a suitable control cell, is indicativeof the test agent being an inhibitor of tumorigenesis.

In another broad aspect, there is provided herein a method ofidentifying an inhibitor of tumorigenesis, comprising providing a testagent to a cell and measuring the level of at least one miR gene productassociated with an altered expression level in a colon cancer-relateddisease, wherein a decrease in the level of the miR gene product in thecell, relative to a suitable control cell, is indicative of the testagent being an inhibitor of tumorigenesis.

In another broad aspect, there is provided herein a marker for assessingone or more metabolic pathways that contribute to at least one ofinitiation, progression, severity, pathology, aggressiveness, grade,activity, disability, mortality, morbidity, disease sub-classificationor other underlying pathogenic or pathological feature of at least onecolon cancer-related disease, wherein the marker comprises one or moremiR gene products selected from the group consisting of miR-20a, miR-21,miR-106a, miR-181b, miR-203 and combinations thereof.

In another broad aspect, there is provided herein a compositioncomprising one or more of the markers described herein.

In another broad aspect, there is provided herein a method ofidentifying a potential for the initiation or development of at leastone colon cancer-related disease in a subject, the method providingmeasuring one or more of the markers described herein. In certainembodiments, one or more markers are present in an isolated sample andall method steps are performed in vitro.

In another broad aspect, there is provided herein a reagent for testingfor a colon cancer-related disease, wherein the reagent comprises apolynucleotide comprising the nucleotide sequence of at least one markerdescribed herein or a nucleotide sequence complementary to thenucleotide sequence of the marker.

In another broad aspect, there is provided herein a reagent for testingfor a colon cancer-related disease, wherein the reagent comprises anantibody that recognizes a protein encoded by at least one markerdescribed herein.

In another broad aspect, there is provided herein a DNA chip for testingfor a colon cancer-related disease, on which a probe has beenimmobilized to assay at least one marker described herein.

In another broad aspect, there is provided herein a method of assessingthe effectiveness of a therapy to prevent, diagnose and/or treat atleast one colon cancer-related disease comprising:

1) subjecting an animal to a therapy whose effectiveness is beingassessed, and

2) determining the level of effectiveness of the treatment being testedin treating or preventing the colon cancer-related disease by evaluatingat least one marker described herein.

In certain embodiments, the candidate therapeutic agent comprises one ormore of: pharmaceutical compositions, nutraceutical compositions, andhomeopathic compositions. Also, the therapy being assessed can be foruse in a human subject. In certain embodiments, the method is not amethod of treatment of the human or animal body by surgery or therapy.

In another broad aspect, there is provided herein a method of assessingthe potential of at least one material for an ability to initiate acolon cancer-related disease response in an animal model, the methodproviding:

1) measuring one or more of up- or down-regulated markers describedherein after exposure of the animal to one or more materials in amountssufficient to initiate a colon cancer-related disease response in theanimal; and

2) determining whether at least one of the up- or down-regulated markershas the ability to initiate a colon cancer-related disease response.

In another broad aspect, there is provided herein a pharmaceuticalcomposition for treating a colon cancer-related disease, comprising: atleast one miR gene product selected from the group consisting ofmiR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof;and, a pharmaceutically-acceptable carrier.

In another broad aspect, there is provided herein a pharmaceuticalcomposition for treating a colon cancer, comprising at least one miRexpression-inhibition compound and a pharmaceutically-acceptablecarrier, wherein the at least one miR expression-inhibition compound isspecific for a miR gene product selected from the group consisting ofmiR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof.

In another broad aspect, there is provided herein an article ofmanufacture comprising: at least one capture reagent that binds to amarker for a colon cancer-related disease selected from at least one ofthe markers described herein.

In another broad aspect, there is provided herein a kit for screeningfor a candidate compound for a therapeutic agent to treat a coloncancer-related disease, wherein the kit comprises: one or more reagentsof at least one marker described herein, and a cell expressing at leastone marker. In certain embodiments, the presence of the marker isdetected using a reagent comprising an antibody or an antibody fragmentwhich specifically binds with at least one marker. Also, in certainembodiments, the reagent is labeled, radio-labeled, or biotin-labeled,and/or the antibody or antibody fragment is radio-labeled,chromophore-labeled, fluorophore-labeled, or enzyme-labeled. In aparticular embodiment, the kit further includes a container comprisingat least one of the markers. Also, the reagent can comprise one or moreof: an antibody, a probe to which the reagent is attached or isattachable, and an immobilized metal chelate.

In another broad aspect, there is provided herein a screening test for acolon cancer-related disease comprising: contacting one or more of themarkers with a substrate for such marker and with a test agent, anddetermining whether the test agent modulates the activity of the marker.

In certain embodiments, all method steps can be performed in vitro.

In another broad aspect, there is provided herein a microarray forpredicting the presence of a colon cancer-related disease in a subjectcomprising an antibody directed to at least one marker.

In another broad aspect, there is provided herein methods, compositionsand the like, where a level of expression of the marker is assessed bydetecting the presence of a transcribed polynucleotide or portionthereof, wherein the transcribed polynucleotide comprises a codingregion of the marker. Also, the sample can be a colon cancer-associatedbody fluid or tissue. In a particular embodiment, the sample comprisescells obtained from the patient.

In another broad aspect, there is provided herein a method for treating,preventing, reversing or limiting the severity of a colon cancer-relateddisease complication in an individual in need thereof, comprising:administering to the individual an agent that interferes with at leastone colon cancer-related disease response signaling pathway, in anamount sufficient to interfere with such signaling, wherein the agentcomprises at least one miR gene product selected from the groupconsisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203 andcombinations thereof.

In another broad aspect, there is provided herein the use of an agentthat interferes with at least one colon cancer-related disease responsesignaling pathway, for the manufacture of a medicament for treating,preventing, reversing or limiting the severity of a colon cancer-relateddisease complication in an individual, wherein the agent comprises atleast one miR gene product selected from the group consisting ofmiR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof.

In another broad aspect, there is provided herein a method of treating,preventing, reversing or limiting the severity of a colon cancer-relateddisease complication in an individual in need thereof, comprisingadministering to the individual an agent that interferes with at leastone colon cancer-related disease response cascade, wherein the agentcomprises at least one miR gene product selected from the groupconsisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203 andcombinations thereof.

In another broad aspect, there is provided herein the use of an agentthat interferes with at least one colon cancer-related disease responsecascade, for the manufacture of a medicament for treating, preventing,reversing or limiting the severity of a colon cancer-related diseasecomplication in an individual, wherein the agent comprises at least onemiR gene product selected from the group consisting of miR-20a, miR-21,miR-106a, miR-181b, miR-203 and combinations thereof.

In another broad aspect, there is provided herein a computer-readablemedium comprising a database having a plurality of digitally-encodedreference profiles, wherein at least a first reference profilerepresents a level of at least a first marker in one or more samplesfrom one or more subjects exhibiting an indicia of a coloncancer-related disease response, wherein the marker comprises one ormore miR gene products selected from the group consisting of miR-20a,miR-21, miR-106a, miR-181b, miR-203 and combinations thereof.

In certain embodiments, the computer readable medium includes at least asecond reference profile that represents a level of at least a secondmarker in one or more samples from one or more subjects exhibitingindicia of a colon cancer-related disease response; or subjects having acolon cancer-related disease.

In another broad aspect, there is provided herein a computer system fordetermining whether a subject has, is predisposed to having, or has apoor survival prognosis for, a colon cancer-related disease, comprisingthe database described herein, and a server comprising acomputer-executable code for causing the computer to receive a profileof a subject, identify from the database a matching reference profilethat is diagnostically relevant to the subject profile, and generate anindication of whether the subject has, or is predisposed to having, acolon cancer-related disease.

In another broad aspect, there is provided herein a computer-assistedmethod for evaluating the presence, absence, nature or extent of a coloncancer-related disease in a subject, comprising:

1) providing a computer comprising a model or algorithm for classifyingdata from a sample obtained from the subject, wherein the classificationincludes analyzing the data for the presence, absence or amount of atleast one marker, wherein the marker comprises one or more miR geneproducts selected from the group consisting of miR-20a, miR-21,miR-106a, miR-181b, miR-203 and combinations thereof;

2) inputting data from the biological sample obtained from the subject;and,

3) classifying the biological sample to indicate the presence, absence,nature or extent of a colon cancer-related disease.

In another broad aspect, at least one miR gene product and combinationsthereof includes isolated variants or biologically-active fragments.

In another broad aspect, there is provided herein an animal model forcolon cancer wherein at least one of the following biological orchemical processes occurs in the animal model up- or down regulation ofone or more miR gene products is selected from the group consisting ofmiR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof.In certain embodiments, the animal model is a nonhuman vertebrate. Inparticular embodiments, the animal model is a mouse, rat, rabbit, orprimate.

Various objects and advantages of this invention will become apparent tothose skilled in the art from the following detailed description of thepreferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1 a-1 g: MiR-21 is expressed at higher levels in colonadenocarcinomas with increasing expression in more advanced tumors.

(FIG. 1 a) In situ hybridization for miR-21 was optimized to distinguishhigh and low expression of miR-21. Colonic epithelial cells in humantumor (T) express higher levels of miR-21 compared to adjacentnontumorous tissue (N). (FIG. 1 c) Nuclei and cytoplasm of colonicepithelial cells in tumor tissue express significant amounts of miR-21in tumor tissue, at high magnification. (FIG. 1 e) Non-tumor tissueshows no significant expression of miR-21 at the same magnification.

(FIG. 1 b, FIG. 1 d, FIG. 1 f) The scramble control probe shows nosignificant staining at low or high magnification in serial sections oftumor and non-tumor tissue, as expected. Scale bars (FIGS. 1 c-f)indicate 500 μM (g) miR-21 is expressed at higher levels in moreadvanced tumors. Dot plots represent miR-21 relative Ct values (fromquantitative RT-PCR) for adenoma and tumor expression levels that havebeen normalized to paired non-adenoma or nontumorous tissue,respectively. Tissue types have been ordered from adenoma to stage I-IVtumors. Bars indicate median value. There is a significant trend thatmore advanced tumors have higher expression of miR-21 (nonparametrictest for trend across ordered groups).

FIG. 2: miR-21 is expressed at higher levels in more advanced tumors.MicroRNA microarrays were used to measure miR-21 expression levels. Dotplots represent miR-21 log₂ (tumor/nontumor ratios) as calculated frommicroRNA microarrays from the original cohort. The probe hsa-miR-21-prec17No 1 from the microarray was used to measure miR-21 expression.Tissues with undetectable expression of miR-21 based on microarray datawere excluded. Tissue types have been ordered from TNM stage I to stageIV tumors. Bars indicate median value. There is a significant trend thatmore advanced tumors have higher expression of miR-21 (p=0.04;nonparametric test for trend across ordered groups).

FIGS. 3 a and 3 b: High miR-21 expression in tumors predicts a poorsurvival in subjects with typical adenocarcinoma histology in bothindependent cohorts. This analysis excludes subjects with eithermucinous adenocarcinoma or adenosquamous carcinoma histology.

(FIG. 3 a) MicroRNA microarrays were used in the Maryland test cohort tomeasure microRNA expression levels of tumors and nontumorous tissues.Tissues with undetectable expression of miR-21 based on microarray datawere excluded. High miR-21 expression was classified based on highesttertile. Red lines indicate individuals with high expression while greenlines correspond to low expression. For nontumorous tissue, 24/69tissues were classified as high while 26/72 tumors were classified ashigh. High miR-21 expression in tumors (right) is associated with poorsurvival while it is not associated in nontumorous tissue.

(FIG. 3 b) Validation of the association with high miR-21 expression intumors and poor prognosis in an independent cohort. Expression levels ofmiR-21 were measured by quantitative RT-PCR. High expression is based onthe highest tertile. 35/103 nontumorous tissues were classified as highand 34/103 tumor tissues were classified as high. P-values are log rankp-values from Kaplan-Meier analysis. X's on all lines indicate the timeat which an individual was censored.

FIGS. 4 a and 4 b: High miR-21 expression in tumors predicts poorsurvival in both independent cohorts. This analysis includes allsubjects regardless of adenocarcinoma histology.

(FIG. 4 a) MicroRNA microarrays were used in the Maryland test cohort tomeasure microRNA expression levels of tumors and nontumorous tissues.Tissues with undetectable expression of miR-21 based on microarray datawere excluded. High miR21 expression was classified based on highesttertile. Red lines indicate individuals with high expression while greenlines correspond to low expression. For nontumorous tissue, 26/74tissues were classified as high while 28/79 tumors were classified ashigh. High miR-21 expression in tumors (right) is associated with poorsurvival while it is not associated in nontumorous tissue.

(FIG. 4 b) Validation of the association with high miR-21 expression intumors and poor prognosis in an independent cohort. Expression levels ofmiR-21 were measured by quantitative RT-PCR. High expression is based onthe highest tertile. 37/111 nontumorous tissues were classified as highand 37/111 tumor tissues were classified as high. All p-values are logrank p-values from Kaplan-Meier analysis. X's on all lines indicate thetime at which an individual was censored.

FIGS. 5 a, 5 b and 5 c: High miR-21 expression is associated with a poorresponse to adjuvant chemotherapy for cases with conventionaladenocarcinoma histology. This analysis includes subjects from thevalidation cohort, excluding subjects with mucinous adenocarcinoma oradenosquamous carcinoma histologies.

(FIG. 5 a) Comparison of survival rates for TNM stage II/III subjectswith conventional adenocarcinoma histology by miR-21 expression levelsand receipt of adjuvant chemotherapy. For the 77 stage II/III subjects,25 were classified as low miR-21 receiving therapy, 28 as low miR-21 andnot receiving therapy, 11 as high miR-21 receiving therapy, and 13 ashigh miR-21 and not receiving therapy. For stage II/III subjects whoreceived adjuvant chemotherapy, high miR-21 expression in tumors isassociated with a poor survival (p=0.03).

(FIG. 5 b) Comparison of TNM stage II subjects with conventionaladenocarcinoma histology. For the 33 stage II subjects, 8 wereclassified as low miR-21 receiving therapy, 15 as low miR-21 and notreceiving therapy, 3 as high miR-21 receiving therapy, and 7 as highmiR-21 and not receiving therapy. All stage II subjects who receivedchemotherapy survived for the duration of this study.

(FIG. 5 c) Comparison of TNM stage III subjects with conventionaladenocarcinoma histology. For the 44 stage III subjects, 17 wereclassified as low miR-21 receiving therapy, 13 as low miR-21 and notreceiving therapy, 8 as high miR-21 receiving therapy, and 6 as highmiR-21 and not receiving therapy. For stage III subjects who receivedadjuvant chemotherapy, high miR-21 expression in tumors is associatedwith a poor survival (p=0.02). X's on all lines indicate the time atwhich an individual was censored.

FIGS. 6 a, 6 b and 6 c: Combined analysis of Maryland test cohort andHong Kong validation cohort examining associations between miR-21expression in tumors and receipt of adjuvant chemotherapy withprognosis. This analysis includes all TNM stage II/III subjects fromboth cohorts. Excluded were individuals with mucinous adenocarcinoma oradenosquamous carcinoma histologies. The left column includesKaplan-Meier plots analyzing the association between receipt of adjuvanttherapy and prognosis. The center column includes analysis of theassociation between high miR-21 expression in tumors and prognosis, andthe right column subdivides individuals based on both chemotherapy andmiR-21 expression status.

(FIG. 6 a) All TNM stage II/III subjects. For the 119 stage II/IIIsubjects, 40 were classified as low miR-21 receiving therapy, 41 as lowmiR-21 and not receiving therapy, 16 as high miR-21 receiving therapy,and 22 as high miR-21 and not receiving therapy. High miR-21 expressionis associated with a poor survival for those who receive chemotherapy(p=0.003) as well as those who do not receive therapy (p=0.04).

(FIG. 6 b) All TNM stage II subjects. For the 52 stage II/III subjects,10 were classified as low miR-21 receiving therapy, 25 as low miR-21 andnot receiving therapy, 4 as high miR-21 receiving therapy, and 13 ashigh miR-21 and not receiving therapy. Associations between high miR-21expression and prognosis was not statistically significant inindividuals who received chemotherapy (p=0.11) or those who did notreceive chemotherapy (p=0.06).

(FIG. 6 c) All TNM stage III subjects. For the 67 stage III subjects, 30were classified as low miR-21 receiving therapy, 16 as low miR-21 andnot receiving therapy, 12 as high miR-21 receiving therapy, and 9 ashigh miR-21 and not receiving therapy. High miR-21 expression issignificantly associated with poor survival in stage III subjects whoreceived chemotherapy (p=0.007), but not in subjects who did not receivechemotherapy (p=0.30). X's on all lines indicate the time at which anindividual was censored.

FIGS. 7 a-7 c. Global miRNA profiles are associated with clinical TNMstaging and survival prognosis. Hierarchical clustering of miRNA TINratios resulted in forming two groups arbitrarily named group A andgroup B. The resulting HEAT map and cluster assignments are shown inFIG. 7 a. These two groups were composed of individuals withsignificantly different survival prognoses for TNM staging with group Bindividuals more likely to be diagnosed as either stage III or IVcompared to group A individuals (FIG. 7 b). Kaplan-Meier analysis showsthat Group B individuals also have a worse survival prognosis (FIG. 7c).

FIGS. 8 a-8 i. TIN ratios of individual miRNAs are predictive ofsurvival prognosis. Displayed here are graphs showing TIN ratios by TNMstaging (left) and Kaplan-Meier analysis (right) for each of these 9miRNAs. The Y axis (TIN ratio by TNM staging graphs) indicates thelog(2) transformed TIN ratio for each individual while the Y axis groupsindividuals by TNM staging (I, II, III or IV). The significance valuesshown are the result of a nonparametric test for trend of average TINratio values across individuals grouped by staging. Kaplan-Meier plotsinclude all individuals with TIN ratio data for that particular miRNA.We found that TIN ratios were associated with both clinical staging andsurvival prognosis.

FIGS. 9 a and 9 b. A miRNA signature of 9 miRNAs predicts risk of dyingof colon cancer. TIN ratios of miR-21, miR-106a, miR181 b, miR-16b,miR-203, let-7g, miR-29a, miR-103-2 and miR-10a were each shown to bepredictive of colon cancer prognosis. Hierarchical clustering of TINratios of these 9 miRNAs resulted in dividing individuals into twogroups (1A) with significantly different survival prognoses (1B). GroupB individuals were at a significantly higher risk for dying from coloncancer than group A. Individuals were excluded from this analysis ifthey were missing greater than 2 of the 9 TIN ratios making up the miRNAsignature.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In one broad aspect, there is provided herein the identification ofparticular microRNAs whose expression is altered in cancer cellsassociated with different colon cancers, relative to normal controlcells.

As used herein interchangeably, a “miR gene product,” “microRNA,” “miR,”or “miRNA” refers to the unprocessed (e.g., precursor) or processed(e.g., mature) RNA transcript from a miR gene. As the miR gene productsare not translated into protein, the term “miR gene products” does notinclude proteins. The unprocessed miR gene transcript is also called a“miR precursor” or “miR prec” and typically comprises an RNA transcriptof about 70-100 nucleotides in length. The miR precursor can beprocessed by digestion with an RNAse (for example, Dicer, Argonaut, orRNAse III (e.g., E. coli RNAse III)) into an active 19-25 nucleotide RNAmolecule. This active 19-25 nucleotide RNA molecule is also called the“processed” miR gene transcript or “mature” miRNA.

The active 19-25 nucleotide RNA molecule can be obtained from the miRprecursor through natural processing routes (e.g., using intact cells orcell lysates) or by synthetic processing routes (e.g., using isolatedprocessing enzymes, such as isolated Dicer, Argonaut, or RNAse III). Itis understood that the active 19-25 nucleotide RNA molecule can also beproduced directly by biological or chemical synthesis, without havingbeen processed from the miR precursor. When a microRNA is referred toherein by name, the name corresponds to both the precursor and matureforms, unless otherwise indicated.

In one aspect, there is provided herein methods of diagnosing whether asubject has, or is at risk for developing, a colon cancer, comprisingmeasuring the level of at least one miR gene product in a test samplefrom the subject and comparing the level of the miR gene product in thetest sample to the level of a corresponding miR gene product in acontrol sample. As used herein, a “subject” can be any mammal that has,or is suspected of having, a solid cancer. In a preferred embodiment,the subject is a human who has, or is suspected of having, a coloncancer.

In one embodiment, the at least one miR gene product measured in thetest sample is selected from the group consisting of miR-20a, miR-21,miR-106a, miR-181b, miR-203 and combinations thereof. In a particularembodiment, the miR gene product is miR-21.

The colon cancer-related disease can be any disorder or cancer thatarises from the colon tissues. Such cancers are typically associatedwith the formation and/or presence of tumor masses and can be, forexample, adenocarcinomas.

In one embodiment, the colon is an adenocarcinoma and the at least onemiR gene product measured in the test sample is selected from the groupconsisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203 andcombinations thereof.

In a further embodiment, the at least one miR gene product measured inthe test sample is miR-21.

The level of at least one miR gene product can be measured in abiological sample (e.g., cells, tissues) obtained from the subject. Forexample, a tissue sample (e.g., from a tumor) can be removed from asubject suspected of having a colon cancer-related disease byconventional biopsy techniques. In another embodiment, a blood samplecan be removed from the subject, and blood cells (e.g., white bloodcells) can be isolated for DNA extraction by standard techniques. Theblood or tissue sample is preferably obtained from the subject prior toinitiation of radiotherapy, chemotherapy or other therapeutic treatment.A corresponding control tissue or blood sample can be obtained fromunaffected tissues of the subject, from a normal human individual orpopulation of normal individuals, or from cultured cells correspondingto the majority of cells in the subject's sample. The control tissue orblood sample is then processed along with the sample from the subject,so that the levels of miR gene product produced from a given miR gene incells from the subject's sample can be compared to the corresponding miRgene product levels from cells of the control sample. A reference miRexpression standard for the biological sample can also be used as acontrol.

An alteration (e.g., an increase or decrease) in the level of a miR geneproduct in the sample obtained from the subject, relative to the levelof a corresponding miR gene product in a control sample, is indicativeof the presence of a colon cancer-related disease in the subject.

In one embodiment, the level of the at least one miR gene product in thetest sample is greater than the level of the corresponding miR geneproduct in the control sample (i.e., expression of the miR gene productis “up-regulated”). As used herein, expression of a miR gene product is“up-regulated” when the amount of miR gene product in a cell or tissuesample from a subject is greater than the amount of the same geneproduct in a control cell or tissue sample.

In another embodiment, the level of the at least one miR gene product inthe test sample is less than the level of the corresponding miR geneproduct in the control sample (i.e., expression of the miR gene productis “down-regulated”). As used herein, expression of a miR gene is“down-regulated” when the amount of miR gene product produced from thatgene in a cell or tissue sample from a subject is less than the amountproduced from the same gene in a control cell or tissue sample.

The relative miR gene expression in the control and normal samples canbe determined with respect to one or more RNA expression standards. Thestandards can comprise, for example, a zero miR gene expression level,the miR gene expression level in a standard cell line, the miR geneexpression level in unaffected tissues of the subject, or the averagelevel of miR gene expression previously obtained for a population ofnormal human controls.

The level of a miR gene product in a sample can be measured using anytechnique that is suitable for detecting RNA expression levels in abiological sample. Suitable techniques (e.g., Northern blot analysis,RT-PCR, in situ hybridization) for determining RNA expression levels ina biological sample (e.g., cells, tissues) are well known to those ofskill in the art. In a particular embodiment, the level of at least onemiR gene product is detected using Northern blot analysis. For example,total cellular RNA can be purified from cells by homogenization in thepresence of nucleic acid extraction buffer, followed by centrifugation.Nucleic acids are precipitated, and DNA is removed by treatment withDNase and precipitation. The RNA molecules are then separated by gelelectrophoresis on agarose gels according to standard techniques, andtransferred to nitrocellulose filters. The RNA is then immobilized onthe filters by heating. Detection and quantification of specific RNA isaccomplished using appropriately labeled DNA or RNA probes complementaryto the RNA in question. See, for example, Molecular Cloning: ALaboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold SpringHarbor Laboratory Press, 1989, Chapter 7, the entire disclosure of whichis incorporated by reference.

Suitable probes for Northern blot hybridization of a given miR geneproduct can be produced from the nucleic acid sequences and include, butare not limited to, probes having at least about 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% or complete complementarity to a miR gene product ofinterest. Methods for preparation of labeled DNA and RNA probes, and theconditions for hybridization thereof to target nucleotide sequences, aredescribed in Molecular Cloning: A Laboratory Manual, J. Sambrook et al.,eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapters10 and 11, the disclosures of which are incorporated herein byreference.

In one non-limiting example, the nucleic acid probe can be labeled with,e.g., a radionuclide, such as ³H, ³²P, ³³P, ¹⁴C, or ³⁵S; a heavy metal;a ligand capable of functioning as a specific binding pair member for alabeled ligand (e.g., biotin, avidin or an antibody); a fluorescentmolecule; a chemiluminescent molecule; an enzyme or the like.

Probes can be labeled to high specific activity by either the nicktranslation method of Rigby et al. (1977), J. Mol. Biol. 113:237-251 orby the random priming method of Fienberg et al. (1983), Anal. Biochem.132:6-13, the entire disclosures of which are incorporated herein byreference. The latter is the method of choice for synthesizing³²P-labeled probes of high specific activity from single-stranded DNA orfrom RNA templates. For example, by replacing preexisting nucleotideswith highly radioactive nucleotides according to the nick translationmethod, it is possible to prepare ³²P-labeled nucleic acid probes with aspecific activity well in excess of 10⁸ cpm/microgram. Autoradiographicdetection of hybridization can then be performed by exposing hybridizedfilters to photographic film. Densitometric scanning of the photographicfilms exposed by the hybridized filters provides an accurate measurementof miR gene transcript levels. Using another approach, miR genetranscript levels can be quantified by computerized imaging systems,such as the Molecular Dynamics 400-B 2D Phosphorimager available fromAmersham Biosciences, Piscataway, N.J.

Where radionuclide labeling of DNA or RNA probes is not practical, therandom-primer method can be used to incorporate an analogue, forexample, the dTTP analogue5-(N—(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridinetriphosphate, into the probe molecule. The biotinylated probeoligonucleotide can be detected by reaction with biotin-bindingproteins, such as avidin, streptavidin, and antibodies (e.g.,anti-biotin antibodies) coupled to fluorescent dyes or enzymes thatproduce color reactions.

In addition to Northern and other RNA hybridization techniques,determining the levels of RNA transcripts can be accomplished using thetechnique of in situ hybridization. This technique requires fewer cellsthan the Northern blotting technique, and involves depositing wholecells onto a microscope cover slip and probing the nucleic acid contentof the cell with a solution containing radioactive or otherwise labelednucleic acid (e.g., cDNA or RNA) probes. This technique is particularlywell-suited for analyzing tissue biopsy samples from subjects. Thepractice of the in situ hybridization technique is described in moredetail in U.S. Pat. No. 5,427,916, the entire disclosure of which isincorporated herein by reference.

In one non-limiting example, suitable probes for in situ hybridizationof a given miR gene product can be produced from the nucleic acidsequences, and include, but are not limited to, probes having at leastabout 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or complete complementarityto a miR gene product of interest, as described above.

The relative number of miR gene transcripts in cells can also bedetermined by reverse transcription of miR gene transcripts, followed byamplification of the reverse-transcribed transcripts by polymerase chainreaction (RT-PCR). The levels of miR gene transcripts can be quantifiedin comparison with an internal standard, for example, the level of mRNAfrom a “housekeeping” gene present in the same sample. A suitable“housekeeping” gene for use as an internal standard includes, e.g.,myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Methods forperforming quantitative and semi-quantitative RT-PCR, and variationsthereof, are well known to those of skill in the art.

In some instances, it may be desirable to simultaneously determine theexpression level of a plurality of different miR gene products in asample. In other instances, it may be desirable to determine theexpression level of the transcripts of all known miR genes correlatedwith a cancer. Assessing cancer-specific expression levels for hundredsof miR genes or gene products is time consuming and requires a largeamount of total RNA (e.g., at least 20 μg for each Northern blot) andautoradiographic techniques that require radioactive isotopes.

To overcome these limitations, an oligolibrary, in microchip format(i.e., a microarray), may be constructed containing a set ofoligonucleotide (e.g., oligodeoxynucleotides) probes that are specificfor a set of miR genes. Using such a microarray, the expression level ofmultiple microRNAs in a biological sample can be determined by reversetranscribing the RNAs to generate a set of target oligodeoxynucleotides,and hybridizing them to probe the oligonucleotides on the microarray togenerate a hybridization, or expression, profile. The hybridizationprofile of the test sample can then be compared to that of a controlsample to determine which microRNAs have an altered expression level insolid cancer cells.

As used herein, “probe oligonucleotide” or “probe oligodeoxynucleotide”refers to an oligonucleotide that is capable of hybridizing to a targetoligonucleotide. “Target oligonucleotide” or “targetoligodeoxynucleotide” refers to a molecule to be detected (e.g., viahybridization). By “miR-specific probe oligonucleotide” or “probeoligonucleotide specific for a miR” is meant a probe oligonucleotidethat has a sequence selected to hybridize to a specific miR geneproduct, or to a reverse transcript of the specific miR gene product.

An “expression profile” or “hybridization profile” of a particularsample is essentially a fingerprint of the state of the sample; whiletwo states may have any particular gene similarly expressed, theevaluation of a number of genes simultaneously allows the generation ofa gene expression profile that is unique to the state of the cell. Thatis, normal tissue may be distinguished from cancerous (e.g., tumor)tissue, and within cancerous tissue, different prognosis states (forexample, good or poor long term survival prospects) may be determined.By comparing expression profiles of the colon cancer tissue in differentstates, information regarding which genes are important (including bothup- and down-regulation of genes) in each of these states is obtained.The identification of sequences that are differentially expressed incolon cancer tissue, as well as differential expression resulting indifferent prognostic outcomes, allows the use of this information in anumber of ways.

In one non-limiting example, a particular treatment regime may beevaluated (e.g., to determine whether a chemotherapeutic drug acts toimprove the long-term prognosis in a particular patient). Similarly,diagnosis may be done or confirmed by comparing patient samples withknown expression profiles. Furthermore, these gene expression profiles(or individual genes) allow screening of drug candidates that suppressthe colon cancer expression profile or convert a poor prognosis profileto a better prognosis profile.

Accordingly, there is also provided herein methods of diagnosing whethera subject has, or is at risk for developing, a colon cancer, comprisingreverse transcribing RNA from a test sample obtained from the subject toprovide a set of target oligodeoxynucleotides, hybridizing the targetoligodeoxynucleotides to a microarray comprising miRNA-specific probeoligonucleotides to provide a hybridization profile for the test sample,and comparing the test sample hybridization profile to a hybridizationprofile generated from a control sample or reference standard, whereinan alteration in the signal of at least one miRNA is indicative of thesubject either having, or being at risk for developing, a solid cancer.

In one embodiment, the microarray comprises miRNA-specific probeoligonucleotides for a substantial portion of all known human miRNAs. Ina particular embodiment, the microarray comprises miRNA-specific probeoligonucleotides for one or more miRNAs selected from the groupconsisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203 andcombinations thereof.

The microarray can be prepared from gene-specific oligonucleotide probesgenerated from known miRNA sequences. The array may contain twodifferent oligonucleotide probes for each miRNA, one containing theactive, mature sequence and the other being specific for the precursorof the miRNA. The array may also contain controls, such as one or moremouse sequences differing from human orthologs by only a few bases,which can serve as controls for hybridization stringency conditions.tRNAs or other RNAs (e.g., rRNAs, mRNAs) from both species may also beprinted on the microchip, providing an internal, relatively stable,positive control for specific hybridization. One or more appropriatecontrols for non-specific hybridization may also be included on themicrochip. For this purpose, sequences are selected based upon theabsence of any homology with any known miRNAs.

The microarray may be fabricated using techniques known in the art. Forexample, probe oligonucleotides of an appropriate length, e.g., 40nucleotides, are 5′-amine modified at position C6 and printed usingcommercially available microarray systems, e.g., the GeneMachineOmniGrid™ 100 Microarrayer and Amersham CodeLink™ activated slides.Labeled cDNA oligomer corresponding to the target RNAs is prepared byreverse transcribing the target RNA with labeled primer. Following firststrand synthesis, the RNA/DNA hybrids are denatured to degrade the RNAtemplates. The labeled target cDNAs thus prepared are then hybridized tothe microarray chip under hybridizing conditions, e.g., 6×SSPE/30%formamide at 25° C. for 18 hours, followed by washing in 0.75×TNT (TrisHCl/NaCl/Tween 20) at 37° C. for 40 minutes. At positions on the arraywhere the immobilized probe DNA recognizes a complementary target cDNAin the sample, hybridization occurs. The labeled target cDNA marks theexact position on the array where binding occurs, allowing automaticdetection and quantification. The output consists of a list ofhybridization events, indicating the relative abundance of specific cDNAsequences, and therefore the relative abundance of the correspondingcomplementary miRs, in the patient sample.

According to one embodiment, the labeled cDNA oligomer is abiotin-labeled cDNA, prepared from a biotin-labeled primer. Themicroarray is then processed by direct detection of thebiotin-containing transcripts using, e.g., Streptavidin-Alexa647conjugate, and scanned utilizing conventional scanning methods. Imageintensities of each spot on the array are proportional to the abundanceof the corresponding miR in the patient sample.

The use of the array has several advantages for miRNA expressiondetection. First, the global expression of several hundred genes can beidentified in the same sample at one time point. Second, through carefuldesign of the oligonucleotide probes, expression of both mature andprecursor molecules can be identified. Third, in comparison withNorthern blot analysis, the chip requires a small amount of RNA, andprovides reproducible results using 2.5 μg of total RNA. The relativelylimited number of miRNAs (a few hundred per species) allows theconstruction of a common microarray for several species, with distinctoligonucleotide probes for each. Such a tool allows for analysis oftrans-species expression for each known miR under various conditions.

In addition to use for quantitative expression level assays of specificmiRs, a microchip containing miRNA-specific probe oligonucleotidescorresponding to a substantial portion of the miRNome, preferably theentire miRNome, may be employed to carry out miR gene expressionprofiling, for analysis of miR expression patterns. Distinct miRsignatures can be associated with established disease markers, ordirectly with a disease state.

According to the expression profiling methods described herein, totalRNA from a sample from a subject suspected of having a coloncancer-related disease quantitatively reverse transcribed to provide aset of labeled target oligodeoxynucleotides complementary to the RNA inthe sample. The target oligodeoxynucleotides are then hybridized to amicroarray comprising miRNA-specific probe oligonucleotides to provide ahybridization profile for the sample. The result is a hybridizationprofile for the sample representing the expression pattern of miRNA inthe sample. The hybridization profile comprises the signal from thebinding of the target oligodeoxynucleotides from the sample to themiRNA-specific probe oligonucleotides in the microarray. The profile maybe recorded as the presence or absence of binding (signal vs. zerosignal).

More preferably, the profile recorded includes the intensity of thesignal from each hybridization. The profile is compared to thehybridization profile generated from a normal, i.e., noncancerous,control sample. An alteration in the signal is indicative of thepresence of, or propensity to develop, cancer in the subject.

Other techniques for measuring miR gene expression are also within theskill in the art, and include various techniques for measuring rates ofRNA transcription and degradation.

There is also provided herein methods of determining the prognosis of asubject with a colon cancer, comprising measuring the level of at leastone miR gene product, which is associated with a particular prognosis ina colon cancer-related disease (e.g., a good or positive prognosis, apoor or adverse prognosis), in a test sample from the subject.

According to these methods, an alteration in the level of a miR geneproduct that is associated with a particular prognosis in the testsample, as compared to the level of a corresponding miR gene product ina control sample, is indicative of the subject having a solid cancerwith a particular prognosis. In one embodiment, the miR gene product isassociated with an adverse (i.e., poor) prognosis. Examples of anadverse prognosis include, but are not limited to, low survival rate andrapid disease progression. In certain embodiments, the level of the atleast one miR gene product is measured by reverse transcribing RNA froma test sample obtained from the subject to provide a set of targetoligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to amicroarray that comprises miRNA-specific probe oligonucleotides toprovide a hybridization profile for the test sample, and comparing thetest sample hybridization profile to a hybridization profile generatedfrom a control sample.

Without wishing to be bound by any one theory, it is believed thatalterations in the level of one or more miR gene products in cells canresult in the deregulation of one or more intended targets for thesemiRs, which can lead to the formation of solid cancers. Therefore,altering the level of the miR gene product (e.g., by decreasing thelevel of a miR gene product that is up-regulated in solid cancer cells,by increasing the level of a miR gene product that is down-regulated insolid cancer cells) may successfully treat the solid cancer.

Accordingly, there is further provided herein methods of inhibitingtumorigenesis in a subject who has, or is suspected of having, a solidcancer wherein at least one miR gene product is deregulated (e.g.,down-regulated, up-regulated) in the cancer cells of the subject. Whenthe at least one isolated miR gene product is down-regulated in thecancer cells (e.g., miR-21), the method comprises administering aneffective amount of the at least one isolated miR gene product, or anisolated variant or biologically-active fragment thereof, such thatproliferation of cancer cells in the subject is inhibited.

For example, when a miR gene product is down-regulated in a cancer cellin a subject, administering an effective amount of an isolated miR geneproduct to the subject can inhibit proliferation of the cancer cell. Theisolated miR gene product that is administered to the subject can beidentical to the endogenous wild-type miR gene product (e.g., a miR geneproduct) that is down-regulated in the cancer cell or it can be avariant or biologically-active fragment thereof.

As defined herein, a “variant” of a miR gene product refers to a miRNAthat has less than 100% identity to a corresponding wild-type miR geneproduct and possesses one or more biological activities of thecorresponding wild-type miR gene product. Examples of such biologicalactivities include, but are not limited to, inhibition of expression ofa target RNA molecule (e.g., inhibiting translation of a target RNAmolecule, modulating the stability of a target RNA molecule, inhibitingprocessing of a target RNA molecule) and inhibition of a cellularprocess associated with solid cancer (e.g., cell differentiation, cellgrowth, cell death). These variants include species variants andvariants that are the consequence of one or more mutations (e.g., asubstitution, a deletion, an insertion) in a miR gene. In certainembodiments, the variant is at least about 95%, 98%, or 99% identical toa corresponding wild-type miR gene product.

As defined herein, a “biologically-active fragment” of a miR geneproduct refers to an RNA fragment of a miR gene product that possessesone or more biological activities of a corresponding wild-type miR geneproduct. As described above, examples of such biological activitiesinclude, but are not limited to, inhibition of expression of a targetRNA molecule and inhibition of a cellular process associated with acolon cancer. In certain embodiments, the biologically-active fragmentis at least about 15 or 17 nucleotides in length. In a particularembodiment, an isolated miR gene product can be administered to asubject in combination with one or more additional anti-cancertreatments. Suitable anti-cancer treatments include, but are not limitedto, chemotherapy, radiation therapy and combinations thereof (e.g.,chemoradiation).

When the at least one isolated miR gene product is up-regulated in thecancer cells, the method comprises administering to the subject aneffective amount of at least one compound for inhibiting expression ofthe at least one miR gene product, referred to herein as miR geneexpression-inhibition compounds, such that proliferation of solid cancercells is inhibited. In a particular embodiment, the at least one miRexpression-inhibition compound is specific for a miR gene productselected from the group consisting miR-20a, miR-21, miR-106a, miR-181b,miR-203 and combinations thereof.

A miR gene expression-inhibiting compound can be administered to asubject in combination with one or more additional anti-cancertreatments. Suitable anti-cancer treatments include, but are not limitedto, chemotherapy, radiation therapy and combinations thereof (e.g.,chemoradiation).

The terms “treat”, “treating” and “treatment”, as used herein, refer toameliorating symptoms associated with a disease or condition, forexample, a solid cancer, including preventing or delaying the onset ofthe disease symptoms, and/or lessening the severity or frequency ofsymptoms of the disease or condition. The terms “subject”, “patient” and“individual” are defined herein to include animals, such as mammals,including, but not limited to, primates, cows, sheep, goats, horses,dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine,equine, canine, feline, rodent, or murine species. In a preferredembodiment, the animal is a human.

As used herein, an “effective amount” of an isolated miR gene product isan amount sufficient to inhibit proliferation of a cancer cell in asubject suffering from a solid cancer. One skilled in the art canreadily determine an effective amount of a miR gene product to beadministered to a given subject, by taking into account factors, such asthe size and weight of the subject; the extent of disease penetration;the age, health and sex of the subject; the route of administration; andwhether the administration is regional or systemic.

For example, an effective amount of an isolated miR gene product can bebased on the approximate weight of a tumor mass to be treated. Theapproximate weight of a tumor mass can be determined by calculating theapproximate volume of the mass, wherein one cubic centimeter of volumeis roughly equivalent to one gram. An effective amount of the isolatedmiR gene product based on the weight of a tumor mass can be in the rangeof about 10-500 micrograms/gram of tumor mass. In certain embodiments,the tumor mass can be at least about 10 micrograms/gram of tumor mass,at least about 60 micrograms/gram of tumor mass or at least about 100micrograms/gram of tumor mass.

An effective amount of an isolated miR gene product can also be based onthe approximate or estimated body weight of a subject to be treated.Preferably, such effective amounts are administered parenterally orenterally, as described herein. For example, an effective amount of theisolated miR gene product is administered to a subject can range fromabout 5 to about 3000 micrograms/kg of body weight, from about 700-1000micrograms/kg of body weight, or greater than about 1000 micrograms/kgof body weight.

One skilled in the art can also readily determine an appropriate dosageregimen for the administration of an isolated miR gene product to agiven subject. For example, a miR gene product can be administered tothe subject once (e.g., as a single injection or deposition).Alternatively, a miR gene product can be administered once or twicedaily to a subject for a period of from about three to abouttwenty-eight days, more particularly from about seven to about ten days.In a particular dosage regimen, a miR gene product is administered oncea day for seven days. Where a dosage regimen comprises multipleadministrations, it is understood that the effective amount of the miRgene product administered to the subject can comprise the total amountof gene product administered over the entire dosage regimen.

As used herein, an “isolated” miR gene product is one that issynthesized, or altered or removed from the natural state through humanintervention. For example, a synthetic miR gene product, or a miR geneproduct partially or completely separated from the coexisting materialsof its natural state, is considered to be “isolated.” An isolated miRgene product can exist in substantially-purified form, or can exist in acell into which the miR gene product has been delivered. Thus, a miRgene product that is deliberately delivered to, or expressed in, a cellis considered an “isolated” miR gene product. A miR gene productproduced inside a cell from a miR precursor molecule is also consideredto be an “isolated” molecule. According to one particular embodiment,the isolated miR gene products described herein can be used for themanufacture of a medicament for treating a solid cancer in a subject(e.g., a human).

Isolated miR gene products can be obtained using a number of standardtechniques. For example, the miR gene products can be chemicallysynthesized or recombinantly produced using methods known in the art. Inone embodiment, miR gene products are chemically synthesized usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include, e.g., Proligo (Hamburg,Germany), Dharmacon Research (Lafayette, Colo., U.S.A.), Pierce Chemical(part of Perbio Science, Rockford, Ill., U.S.A.), Glen Research(Sterling, Va., U.S.A.), ChemGenes (Ashland, Mass., U.S.A.) and Cruachem(Glasgow, UK).

Alternatively, the miR gene products can be expressed from recombinantcircular or linear DNA plasmids using any suitable promoter. Suitablepromoters for expressing RNA from a plasmid include, e.g., the U6 or H1RNA pol III promoter sequences, or the cytomegalovirus promoters.Selection of other suitable promoters is within the skill in the art.The recombinant plasmids of the invention can also comprise inducible orregulatable promoters for expression of the miR gene products in cancercells.

The miR gene products that are expressed from recombinant plasmids canbe isolated from cultured cell expression systems by standardtechniques. The miR gene products that are expressed from recombinantplasmids can also be delivered to, and expressed directly in, the cancercells. The use of recombinant plasmids to deliver the miR gene productsto cancer cells is discussed in more detail below.

The miR gene products can be expressed from a separate recombinantplasmid, or they can be expressed from the same recombinant plasmid. Inone embodiment, the miR gene products are expressed as RNA precursormolecules from a single plasmid, and the precursor molecules areprocessed into the functional miR gene product by a suitable processingsystem, including, but not limited to, processing systems extant withina cancer cell. Other suitable processing systems include, e.g., the invitro Drosophila cell lysate system (e.g., as described in U.S.Published Patent Application No. 2002/0086356 to Tuschl et al., theentire disclosure of which is incorporated herein by reference) and theE. coli RNAse III system (e.g., as described in U.S. Published PatentApplication No. 2004/0014113 to Yang et al., the entire disclosure ofwhich is incorporated herein by reference).

Selection of plasmids suitable for expressing the miR gene products,methods for inserting nucleic acid sequences into the plasmid to expressthe gene products, and methods of delivering the recombinant plasmid tothe cells of interest are within the skill in the art. See, for example,Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat.Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553;Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al.(2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol.20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, theentire disclosures of which are incorporated herein by reference.

In one embodiment, a plasmid expressing the miR gene products comprisesa sequence encoding a miR precursor RNA under the control of the CMVintermediate-early promoter. As used herein, “under the control” of apromoter means that the nucleic acid sequences encoding the miR geneproduct are located 3′ of the promoter, so that the promoter caninitiate transcription of the miR gene product coding sequences.

The miR gene products can also be expressed from recombinant viralvectors. It is contemplated that the miR gene products can be expressedfrom two separate recombinant viral vectors, or from the same viralvector. The RNA expressed from the recombinant viral vectors can eitherbe isolated from cultured cell expression systems by standardtechniques, or can be expressed directly in cancer cells. The use ofrecombinant viral vectors to deliver the miR gene products to cancercells is discussed in more detail below.

The recombinant viral vectors of the invention comprise sequencesencoding the miR gene products and any suitable promoter for expressingthe RNA sequences. Suitable promoters include, but are not limited to,the U6 or H1 RNA pol III promoter sequences, or the cytomegaloviruspromoters. Selection of other suitable promoters is within the skill inthe art. The recombinant viral vectors of the invention can alsocomprise inducible or regulatable promoters for expression of the miRgene products in a cancer cell.

Any viral vector capable of accepting the coding sequences for the miRgene products can be used; for example, vectors derived from adenovirus(AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses(LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.The tropism of the viral vectors can be modified by pseudotyping thevectors with envelope proteins or other surface antigens from otherviruses, or by substituting different viral capsid proteins, asappropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorsthat express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz, I. E., et al. (2002), J. Virol.76:791-801, the entire disclosure of which is incorporated herein byreference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingRNA into the vector, methods of delivering the viral vector to the cellsof interest, and recovery of the expressed RNA products are within theskill in the art. See, for example, Dornburg (1995), Gene Therapy2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum.Gene Therapy 1:5-14; and Anderson (1998), Nature 392:25-30, the entiredisclosures of which are incorporated herein by reference.

Particularly suitable viral vectors are those derived from AV and AAV. Asuitable AV vector for expressing the miR gene products, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia et al. (2002), Nat.Biotech. 20:1006-1010, the entire disclosure of which is incorporatedherein by reference. Suitable AAV vectors for expressing the miR geneproducts, methods for constructing the recombinant AAV vector, andmethods for delivering the vectors into target cells are described inSamulski et al. (1987), J. Virol. 61:3096-3101; Fisher et al. (1996), J.Virol., 70:520-532; Samulski et al. (1989), J. Virol. 63:3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are incorporated herein byreference. In one embodiment, the miR gene products are expressed from asingle recombinant AAV vector comprising the CMV intermediate earlypromoter.

In a certain embodiment, a recombinant AAV viral vector of the inventioncomprises a nucleic acid sequence encoding a miR precursor RNA inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter. As used herein, “in operable connection witha polyT termination sequence” means that the nucleic acid sequencesencoding the sense or antisense strands are immediately adjacent to thepolyT termination signal in the 5′ direction. During transcription ofthe miR sequences from the vector, the polyT termination signals act toterminate transcription.

In other embodiments of the treatment methods of the invention, aneffective amount of at least one compound that inhibits miR expressioncan be administered to the subject. As used herein, “inhibiting miRexpression” means that the production of the precursor and/or active,mature form of miR gene product after treatment is less than the amountproduced prior to treatment. One skilled in the art can readilydetermine whether miR expression has been inhibited in a cancer cell,using, for example, the techniques for determining miR transcript leveldiscussed above for the diagnostic method. Inhibition can occur at thelevel of gene expression (i.e., by inhibiting transcription of a miRgene encoding the miR gene product) or at the level of processing (e.g.,by inhibiting processing of a miR precursor into a mature, active miR).

As used herein, an “effective amount” of a compound that inhibits miRexpression is an amount sufficient to inhibit proliferation of a cancercell in a subject suffering from a cancer (e.g., a colon cancer). Oneskilled in the art can readily determine an effective amount of a miRexpression-inhibition compound to be administered to a given subject, bytaking into account factors, such as the size and weight of the subject;the extent of disease penetration; the age, health and sex of thesubject; the route of administration; and whether the administration isregional or systemic.

For example, an effective amount of the expression-inhibition compoundcan be based on the approximate weight of a tumor mass to be treated, asdescribed herein. An effective amount of a compound that inhibits miRexpression can also be based on the approximate or estimated body weightof a subject to be treated, as described herein.

One skilled in the art can also readily determine an appropriate dosageregimen for administering a compound that inhibits miR expression to agiven subject.

Suitable compounds for inhibiting miR gene expression includedouble-stranded RNA (such as short- or small-interfering RNA or“siRNA”), antisense nucleic acids, and enzymatic RNA molecules, such asribozymes. Each of these compounds can be targeted to a given miR geneproduct and interfere with the expression of (e.g., inhibit translationof, induce cleavage or destruction of) the target miR gene product.

For example, expression of a given miR gene can be inhibited by inducingRNA interference of the miR gene with an isolated double-stranded RNA(“dsRNA”) molecule which has at least 90%, for example at least 95%, atleast 98%, at least 99%, or 100%, sequence homology with at least aportion of the miR gene product. In a particular embodiment, the dsRNAmolecule is a “short or small interfering RNA” or “siRNA.”

siRNA useful in the present methods comprise short double-stranded RNAfrom about 17 nucleotides to about 29 nucleotides in length, preferablyfrom about 19 to about 25 nucleotides in length. The siRNA comprise asense RNA strand and a complementary antisense RNA strand annealedtogether by standard Watson-Crick base-pairing interactions (hereinafter“base-paired”). The sense strand comprises a nucleic acid sequence thatis substantially identical to a nucleic acid sequence contained withinthe target miR gene product.

As used herein, a nucleic acid sequence in an siRNA which is“substantially identical” to a target sequence contained within thetarget mRNA is a nucleic acid sequence that is identical to the targetsequence, or that differs from the target sequence by one or twonucleotides. The sense and antisense strands of the siRNA can comprisetwo complementary, single-stranded RNA molecules, or can comprise asingle molecule in which two complementary portions are base-paired andare covalently linked by a single-stranded “hairpin” area.

The siRNA can also be altered RNA that differs from naturally-occurringRNA by the addition, deletion, substitution and/or alteration of one ormore nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siRNA or to one ormore internal nucleotides of the siRNA, or modifications that make thesiRNA resistant to nuclease digestion, or the substitution of one ormore nucleotides in the siRNA with deoxyribonucleotides.

One or both strands of the siRNA can also comprise a 3′ overhang. Asused herein, a “3′ overhang” refers to at least one unpaired nucleotideextending from the 3′-end of a duplexed RNA strand. Thus, in certainembodiments, the siRNA comprises at least one 3′ overhang of from 1 toabout 6 nucleotides (which includes ribonucleotides ordeoxyribonucleotides) in length, from 1 to about 5 nucleotides inlength, from 1 to about 4 nucleotides in length, or from about 2 toabout 4 nucleotides in length. In a particular embodiment, the 3′overhang is present on both strands of the siRNA, and is 2 nucleotidesin length. For example, each strand of the siRNA can comprise 3′overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).

The siRNA can be produced chemically or biologically, or can beexpressed from a recombinant plasmid or viral vector, as described abovefor the isolated miR gene products. Exemplary methods for producing andtesting dsRNA or siRNA molecules are described in U.S. Published PatentApplication No. 2002/0173478 to Gewirtz and in U.S. Published PatentApplication No. 2004/0018176 to Reich et al., the entire disclosures ofboth of which are incorporated herein by reference.

Expression of a given miR gene can also be inhibited by an antisensenucleic acid. As used herein, an “antisense nucleic acid” refers to anucleic acid molecule that binds to target RNA by means of RNA-RNA,RNA-DNA or RNA-peptide nucleic acid interactions, which alters theactivity of the target RNA. Antisense nucleic acids suitable for use inthe present methods are single-stranded nucleic acids (e.g., RNA, DNA,RNA-DNA chimeras, peptide nucleic acid (PNA)) that generally comprise anucleic acid sequence complementary to a contiguous nucleic acidsequence in a miR gene product. The antisense nucleic acid can comprisea nucleic acid sequence that is 50-100% complementary, 75-100%complementary, or 95-100% complementary to a contiguous nucleic acidsequence in a miR gene product.

Without wishing to be bound by any theory, it is believed that theantisense nucleic acids activate RNase H or another cellular nucleasethat digests the miR gene product/antisense nucleic acid duplex.

Antisense nucleic acids can also contain modifications to the nucleicacid backbone or to the sugar and base moieties (or their equivalent) toenhance target specificity, nuclease resistance, delivery or otherproperties related to efficacy of the molecule. Such modificationsinclude cholesterol moieties, duplex intercalators, such as acridine, orone or more nuclease-resistant groups.

Antisense nucleic acids can be produced chemically or biologically, orcan be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing are within the skill in the art; see, e.g.,Stein and Cheng (1993), Science 261:1004 and U.S. Pat. No. 5,849,902 toWoolf et al., the entire disclosures of which are incorporated herein byreference.

Expression of a given miR gene can also be inhibited by an enzymaticnucleic acid. As used herein, an “enzymatic nucleic acid” refers to anucleic acid comprising a substrate binding region that hascomplementarity to a contiguous nucleic acid sequence of a miR geneproduct, and which is able to specifically cleave the miR gene product.The enzymatic nucleic acid substrate binding region can be, for example,50-100% complementary, 75-100% complementary, or 95-100% complementaryto a contiguous nucleic acid sequence in a miR gene product. Theenzymatic nucleic acids can also comprise modifications at the base,sugar, and/or phosphate groups. An exemplary enzymatic nucleic acid foruse in the present methods is a ribozyme.

The enzymatic nucleic acids can be produced chemically or biologically,or can be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing dsRNA or siRNA molecules are described inWerner and Uhlenbeck (1995), Nucl. Acids Res. 23:2092-96; Hammann et al.(1999), Antisense and Nucleic Acid Drug Dev. 9:25-31; and U.S. Pat. No.4,987,071 to Cech et al, the entire disclosures of which areincorporated herein by reference.

Administration of at least one miR gene product, or at least onecompound for inhibiting miR expression, will inhibit the proliferationof cancer cells in a subject who has a solid cancer. As used herein, to“inhibit the proliferation of a cancer cell” means to kill the cell, orpermanently or temporarily arrest or slow the growth of the cell.Inhibition of cancer cell proliferation can be inferred if the number ofsuch cells in the subject remains constant or decreases afteradministration of the miR gene products or miR geneexpression-inhibition compounds. An inhibition of cancer cellproliferation can also be inferred if the absolute number of such cellsincreases, but the rate of tumor growth decreases.

The number of cancer cells in the body of a subject can be determined bydirect measurement, or by estimation from the size of primary ormetastatic tumor masses. For example, the number of cancer cells in asubject can be measured by immunohistological methods, flow cytometry,or other techniques designed to detect characteristic surface markers ofcancer cells.

The size of a tumor mass can be ascertained by direct visualobservation, or by diagnostic imaging methods, such as X-ray, magneticresonance imaging, ultrasound, and scintigraphy. Diagnostic imagingmethods used to ascertain size of the tumor mass can be employed with orwithout contrast agents, as is known in the art. The size of a tumormass can also be ascertained by physical means, such as palpation of thetissue mass or measurement of the tissue mass with a measuringinstrument, such as a caliper.

The miR gene products or miR gene expression-inhibition compounds can beadministered to a subject by any means suitable for delivering thesecompounds to cancer cells of the subject. For example, the miR geneproducts or miR expression-inhibition compounds can be administered bymethods suitable to transfect cells of the subject with these compounds,or with nucleic acids comprising sequences encoding these compounds.

In one embodiment, the cells are transfected with a plasmid or viralvector comprising sequences encoding at least one miR gene product ormiR gene expression-inhibition compound.

Transfection methods for eukaryotic cells are well known in the art, andinclude, e.g., direct injection of the nucleic acid into the nucleus orpronucleus of a cell; electroporation; liposome transfer or transfermediated by lipophilic materials; receptor-mediated nucleic aciddelivery, bioballistic or particle acceleration; calcium phosphateprecipitation, and transfection mediated by viral vectors.

For example, cells can be transfected with a liposomal transfercompound, e.g., DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate,Boehringer-Mannheim) or an equivalent, such as LIPOFECTIN. The amount ofnucleic acid used is not critical to the practice of the invention;acceptable results may be achieved with 0.1-100 micrograms of nucleicacid/10⁵ cells. For example, a ratio of about 0.5 micrograms of plasmidvector in 3 micrograms of DOTAP per 10⁵ cells can be used.

A miR gene product or miR gene expression-inhibition compound can alsobe administered to a subject by any suitable enteral or parenteraladministration route. Suitable enteral administration routes for thepresent methods include, e.g., oral, rectal, or intranasal delivery.Suitable parenteral administration routes include, e.g., intravascularadministration (e.g., intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissue injection(e.g., peri-tumoral and intra-tumoral injection, intra-retinalinjection, or subretinal injection); subcutaneous injection ordeposition, including subcutaneous infusion (such as by osmotic pumps);direct application to the tissue of interest, for example by a catheteror other placement device (e.g., a retinal pellet or a suppository or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Particularly suitable administration routes are injection,infusion and direct injection into the tumor.

In the present methods, a miR gene product or miR gene productexpression-inhibition compound can be administered to the subject eitheras naked RNA, in combination with a delivery reagent, or as a nucleicacid (e.g., a recombinant plasmid or viral vector) comprising sequencesthat express the miR gene product or miR gene productexpression-inhibition compound. Suitable delivery reagents include,e.g., the Mirus Transit TKO lipophilic reagent; lipofectin;lipofectamine; cellfectin; polycations (e.g., polylysine), andliposomes.

Recombinant plasmids and viral vectors comprising sequences that expressthe miR gene products or miR gene expression-inhibition compounds, andtechniques for delivering such plasmids and vectors to cancer cells, arediscussed herein and/or are well known in the art.

In a particular embodiment, liposomes are used to deliver a miR geneproduct or miR gene expression-inhibition compound (or nucleic acidscomprising sequences encoding them) to a subject. Liposomes can alsoincrease the blood half-life of the gene products or nucleic acids.Suitable liposomes for use in the invention can be formed from standardvesicle-forming lipids, which generally include neutral or negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of factors, such as thedesired liposome size and half-life of the liposomes in the bloodstream. A variety of methods are known for preparing liposomes, forexample, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng.9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369, the entire disclosures of which are incorporated herein byreference.

The liposomes for use in the present methods can comprise a ligandmolecule that targets the liposome to cancer cells. Ligands that bind toreceptors prevalent in cancer cells, such as monoclonal antibodies thatbind to tumor cell antigens, are preferred.

The liposomes for use in the present methods can also be modified so asto avoid clearance by the mononuclear macrophage system (“MMS”) andreticuloendothelial system (“RES”). Such modified liposomes haveopsonization-inhibition moieties on the surface or incorporated into theliposome structure. In a particularly preferred embodiment, a liposomeof the invention can comprise both an opsonization-inhibition moiety anda ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization-inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer that significantly decreases the uptakeof the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is incorporated herein byreference.

Opsonization-inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG orPPG, and PEG or PPG stearate; synthetic polymers, such as polyacrylamideor poly N-vinyl pyrrolidone; linear, branched, or dendrimericpolyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcoholand polyxylitol to which carboxylic or amino groups are chemicallylinked, as well as gangliosides, such as ganglioside GM1. Copolymers ofPEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are alsosuitable. In addition, the opsonization-inhibiting polymer can be ablock copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. Theopsonization-inhibiting polymers can also be natural polysaccharidescontaining amino acids or carboxylic acids, e.g., galacturonic acid,glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid,neuraminic acid, alginic acid, carrageenan; aminated polysaccharides oroligosaccharides (linear or branched); or carboxylated polysaccharidesor oligosaccharides, e.g., reacted with derivatives of carbonic acidswith resultant linking of carboxylic groups. Preferably, theopsonization-inhibiting moiety is a PEG, PPG, or a derivative thereof.Liposomes modified with PEG or PEG-derivatives are sometimes called“PEGylated liposomes.”

The opsonization-inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture, such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Liposomes modified with opsonization-inhibition moieties remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes. Stealth liposomesare known to accumulate in tissues fed by porous or “leaky”microvasculature. Thus, tissue characterized by such microvasculaturedefects, for example, solid tumors, will efficiently accumulate theseliposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., U.S.A.,18:6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation ofthe liposomes in the liver and spleen. Thus, liposomes that are modifiedwith opsonization-inhibition moieties are particularly suited to deliverthe miR gene products or miR gene expression-inhibition compounds (ornucleic acids comprising sequences encoding them) to tumor cells.

The miR gene products or miR gene expression-inhibition compounds can beformulated as pharmaceutical compositions, sometimes called“medicaments,” prior to administering them to a subject, according totechniques known in the art. Accordingly, the invention encompassespharmaceutical compositions for treating a solid cancer. In oneembodiment, the pharmaceutical composition comprises at least oneisolated miR gene product, or an isolated variant or biologically-activefragment thereof, and a pharmaceutically-acceptable carrier. In aparticular embodiment, the at least one miR gene product corresponds toa miR gene product that has a decreased level of expression in solidcancer cells relative to suitable control cells. In certain embodimentsthe isolated miR gene product is selected from the group consisting ofmiR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof.

In other embodiments, the pharmaceutical compositions of the inventioncomprise at least one miR expression-inhibition compound. In aparticular embodiment, the at least one miR gene expression-inhibitioncompound is specific for a miR gene whose expression is greater in coloncancer cells than control cells. In certain embodiments, the miR geneexpression-inhibition compound is specific for one or more miR geneproducts selected from the group consisting of miR-20a, miR-21,miR-106a, miR-181b, miR-203 and combinations thereof.

Pharmaceutical compositions of the present invention are characterizedas being at least sterile and pyrogen-free. As used herein,“pharmaceutical compositions” include formulations for human andveterinary use. Methods for preparing pharmaceutical compositions of theinvention are within the skill in the art, for example as described inRemington's Pharmaceutical Science, 17th ed., Mack Publishing Company,Easton, Pa. (1985), the entire disclosure of which is incorporatedherein by reference.

The present pharmaceutical compositions comprise at least one miR geneproduct or miR gene expression-inhibition compound (or at least onenucleic acid comprising sequences encoding them) (e.g., 0.1 to 90% byweight), or a physiologically-acceptable salt thereof, mixed with apharmaceutically-acceptable carrier. In certain embodiments, thepharmaceutical compositions of the invention additionally comprise oneor more anti-cancer agents (e.g., chemotherapeutic agents). Thepharmaceutical formulations of the invention can also comprise at leastone miR gene product or miR gene expression-inhibition compound (or atleast one nucleic acid comprising sequences encoding them), which areencapsulated by liposomes and a pharmaceutically-acceptable carrier. Inone embodiment, the pharmaceutical composition comprises a miR gene orgene product that is miR-21.

Especially suitable pharmaceutically-acceptable carriers are water,buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronicacid and the like.

In a particular embodiment, the pharmaceutical compositions of theinvention comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them) that is resistant to degradation by nucleases.One skilled in the art can readily synthesize nucleic acids that arenuclease resistant, for example, by incorporating one or moreribonucleotides that is modified at the 2′-position into the miR geneproduct. Suitable 2′-modified ribonucleotides include those modified atthe 2′-position with fluoro, amino, alkyl, alkoxy, and O-allyl.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude, e.g., physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (such as, for example,calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calciumor sodium salts (for example, calcium chloride, calcium ascorbate,calcium gluconate or calcium lactate). Pharmaceutical compositions ofthe invention can be packaged for use in liquid form, or can belyophilized.

For solid pharmaceutical compositions of the invention, conventionalnontoxic solid pharmaceutically-acceptable carriers can be used; forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of the at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them). A pharmaceutical composition for aerosol(inhalational) administration can comprise 0.01-20% by weight,preferably 1%-10% by weight, of the at least one miR gene product or miRgene expression-inhibition compound (or at least one nucleic acidcomprising sequences encoding them) encapsulated in a liposome asdescribed above, and a propellant. A carrier can also be included asdesired; e.g., lecithin for intranasal delivery.

The pharmaceutical compositions of the invention can further compriseone or more anti-cancer agents. In a particular embodiment, thecompositions comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them) and at least one chemotherapeutic agent.Chemotherapeutic agents that are suitable for the methods of theinvention include, but are not limited to, DNA-alkylating agents,anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizingagents, tubulin destabilizing agents, hormone antagonist agents,topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors,CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinaseinhibitors, antisense nucleic acids, triple-helix DNAs, nucleic acidsaptamers, and molecularly-modified viral, bacterial and exotoxic agents.Examples of suitable agents for the compositions of the presentinvention include, but are not limited to, cytidine arabinoside,methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin),cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin,methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine,camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide,oxaliplatin, irinotecan, topotecan, leucovorin, carmustine,streptozocin, CPT-11, taxol, tamoxifen, dacarbazine, rituximab,daunorubicin, 1-β-D-arabinofuranosylcytosine, imatinib, fludarabine,docetaxel, FOLFOX4.

There is also provided herein methods of identifying an inhibitor oftumorigenesis, comprising providing a test agent to a cell and measuringthe level of at least one miR gene product in the cell. In oneembodiment, the method comprises providing a test agent to a cell andmeasuring the level of at least one miR gene product associated withdecreased expression levels in cancer cells. An increase in the level ofthe miR gene product in the cell after the agent is provided, relativeto a suitable control cell (e.g., agent is not provided), is indicativeof the test agent being an inhibitor of tumorigenesis. In a particularembodiment, at least one miR gene product associated with decreasedexpression levels in cancer cells is selected from the group consistingmiR-20a, miR-21, miR-106a, miR-181b, miR-203 and combinations thereof.

In other embodiments, the method comprises providing a test agent to acell and measuring the level of at least one miR gene product associatedwith increased expression levels in cancer cells. A decrease in thelevel of the miR gene product in the cell after the agent is provided,relative to a suitable control cell (e.g., agent is not provided), isindicative of the test agent being an inhibitor of tumorigenesis. In aparticular embodiment, at least one miR gene product associated withincreased expression levels in cancer cells is selected from the groupconsisting of miR-20a, miR-21, miR-106a, miR-181b, miR-203 andcombinations thereof.

Suitable agents include, but are not limited to drugs (e.g., smallmolecules, peptides), and biological macromolecules (e.g., proteins,nucleic acids). The agent can be produced recombinantly, synthetically,or it may be isolated (i.e., purified) from a natural source. Variousmethods for providing such agents to a cell (e.g., transfection) arewell known in the art, and several of such methods are describedhereinabove. Methods for detecting the expression of at least one miRgene product (e.g., Northern blotting, in situ hybridization, RT-PCR,expression profiling) are also well known in the art. Several of thesemethods are also described hereinabove.

The invention will now be illustrated by the following non-limitingexamples.

Example 1 MicroRNA Expression Patterns are Altered in Colon Tumors

We compared microRNA profiles of 84 pairs of colon tumorous and adjacentnontumorous tissues using microRNA microarrays³⁰. These 84 subjects werepatients recruited from the greater Baltimore, Md. area with incidentcolon adenocarcinoma and are referred to as the Maryland test cohort(Table 1).

TABLE 1 Characteristics of Population and Tumors Maryland Hong KongValidation Test Cohort Cohort N = 84 N = 113 Recruitment area Baltimore,Maryland, USA Hong Kong, China Age at enrollment - yr Mean ± SD 64.6 ±10.7 55.8 ± 15 Range 32-87 32-84 Sex - no. (%) Male 66 (79) 56 (50)Female 18 (21) 57 (50) Race - no. (%) White 52 (62) 0 (0) Black 32 (38)0 (0) Asian 0 (0) 113 (100) Tumor location - no. (%) Distal 48 (59) 90(80) Proximal 34 (41) 23 (20) Adenocarcinoma Histology - no. (%)Adenocarcinoma 75 (89) 105 (93)  Mucinous adenocarcinoma  8 (10) 7 (6)Adenosquamous carcinoma 1 (1) 0 (0) Signet ring cell and mucinous 0 (0)1 (1) Adjuvant Chemotherapy2 - no. (%) Received 22 (37) 40 (35) Did notreceive 37 (63) 73 (65) TNM Stage - no. (%) II 29 (34) 37 (33) III 36(43) 48 (42) IV 10 (12) 19 (17) ¹Distal includes tumors located in ordistal to the descending colon. Proximal tumors include tumors in orproximal to the splenic flexure. Tumor location was available for 82subjects in the original cohort and all subjects in the validationcohort. ²Detailed information pertaining to receipt of chemotherapy wasavailable for 59 subjects in the test cohort and all subjects in thevalidation cohort. Chemotherapy was primarily fluorouracil-based (informs of either intravenous 5-fluorouracil or oral drugs includingtegafur with uracil [UFT]) with or without Levamisole or Leucovorin.

Tumor microRNA profiles were distinctly different than nontumorprofiles. Thirty-seven independent microRNAs were found to bedifferentially expressed in tumors (p<0.001 with false discovery rate<0.5%; Table 2.

TABLE 2 Fold Chromosomal Probe mature miR p-value¹ FDR² Change locationMicroRNAs the are Differentially Expressed in Tumors hsa-mir-21No1miR-21  <1e−07  <1e−07 1.7 17q23.2 hsa-mir-021-prec-17No1 miR-21  <1e−07 <1e−07 1.8 17q23.2 hsa-mir-092-prec-13 = 092- miR-92  <1e−07  <1e−071.4 13g31.3 1No2 hsa-mir-222-precNo2 miR-222 1.40E−06 8.05E−05 1.2Xp11.3 hsa-mir-181b-2No1 miR-181b 1.90E−06 8.74E−05 1.2 9q33.3hsa-mir-210-prec mIR-210 1.12E−05 0.00032 1.2 11p15.5 hsa-mir-020-precmiR-20a 2.53E−05 0.00057 1.5 13q31.3 hsa-mir-106-prec-X miR-106a3.30E−05 0.00058 1.4 X26.2 hsa-mir-106aNo1 miR-106a 3.51E−05 0.00058 1.4X26.2 hsa-mir-093-prec-7.1⁼093-1 miR-93 3.52E−05 0.00058 1.2 7q22.1hsa-mir-335No2 miR-335 3.55E−05 0.00058 1.2 7q32.2 hsa-mir-222-precNolmiR-222 4.27E−05 0.00065 1.2 Xpll.3 hsa-mir-338Nol miR-338 5.78E−050.00074 1.1 17q25.3 hsa-mir-133bNo2 miR-133b 6.50E−05 0.00079 1.1 6p12.2hsa-mir-092-prec-X = 092-2 miR-92 7.95E−05 0.00083 1.4 Xq26.2hsa-mir-346Nol miR-346 8.42E−05 0.00084 1.2 10q23.2 hsa-mir-106bNo1miR-106b 0.0002091 0.00178 1.2 7q22.1 hsa-mir-135-2-prec miR-153a0.0002363 0.00194 1.1 12q23.1 hsa-mir-219-lNo2 miR-219 0.0002515 0.001991.3 9q34.11 hsa-mir-34aNo1 miR-34a 0.000265 0.00203 1.1 lp36.22hsa-mir-099b-prec-19No1 miR-99b 0.0003758 0.00259 1.1 19q13.41hsa-mir-185-precNo2 miR-185 0.0003827 0.00259 1.2 22q11.21hsa-mir-223-prec miR-223 0.0004038 0.00265 1.4 Xq12 hsa-mir-211-precNo2miR-211 0.0004338 0.00277 1.1 15q13.3 hsa-mir-135-1-prec miR-135a0.0004648 0.00287 1.1 3p21.1 hsa-mir-127-prec miR-127 0.0004748 0.002871.1 14q32.31 hsa-mir-203-precNol miR-203 0.0004993 0.00294 1.4 14q32.33hsa-mir-212-precNol miR-212 0.0006339 0.00364 1.1 17p13.3hsa-mir-095-prec-4 miR-95 0.0006996 0.00392 1.2 4p16.1hsa-mir-017-precNo2 miR-17-5p 0.0007252 0.00392 1.3 13q31.3 MicroRNAswith reduced Expression in Tumors hsa-mir-342No2 miR-342 4.00E−060.00015 0.9 14q32.2 hsa-mir-192-2/3Nol miR-192 8.70E−06 0.00029 0.711q13.1 hsa-mir-1-2No2 miR-1 2.22E−05 0.00057 0.9 18g11.2 hsa-mir-34bNo2miR-34b 4.78E−05 0.00069 0.8 11q23.1 hsa-mir-215-precNol miR-2155.26E−05 0.00071 0.7 1q41 hsa-mir-192No1 miR-192 7.36E−05 0.00081 0.711q13.1 hsa-mir-301 No2 miR-301 7.44E−05 0.00081 0.7 17q23.2hsa-miR-324-5pNo2 miR-324-5p 1.00E−04 0.00096 0.9 17p13.1hsa-mir-030a-precNo2 miR-30a-3p 0.0001933 0.00171 0.9 6q13 hsa-mir-1-1No2 miR-1 0.0002906 0.00216 0.9 20q13.33 hsa-mir-34cNo2 miR-34c0.0007334 0.00392 0.9 11q23.1 hsa-mir-331 No2 miR-331 0.0008555 0.004460.9 12q22 hsa-mir-148bNo2 miR-148b 0.0008726 0.00446 0.9 12q13.13¹P-values reported are the result of paired class comparison analysis ofmicroRNA expression patterns from 84 pairs colon adenocarcinomas andnontumorus tissue. ²FDR = False Discovery Rate

Twenty-six microRNAs were expressed at higher levels in tumors withmiR-21 enriched the most at 1.8-fold. Global microRNA profilesdistinguish between tumor and paired nontumorous tissue with 89%accuracy using either the 3-nearest neighbors or nearest centroid classprediction algorithms (10-fold cross validation repeated 100 times),suggesting a systematic change in microRNA expression patterns duringtumor formation.

We chose miR-20a, miR-21, miR-106a, miR-181b and miR-203 for validationbased on their expression differences between tumor and pairednontumorous tissue combined with their association to poor survival. Forvalidation, we measured the expression levels of these microRNAs withqRT-PCR in tumor and paired nontumorous tissue from an independentcohort. The validation cohort consists of 113 patients recruited fromHong Kong, China with incident colon cancer (Table 1).

MiR-20a (2.3-fold), miR-21 (2.8-fold), miR106a (2.4-fold), miR-1811b(1.4-fold) and miR-203 (1.8-fold) were all expressed at higher levels intumors (p<0.001, Wilcoxon matched pairs test) (Table 3a).

TABLE 3 MicroRNA Expression in Tumors vs. Paired Nontumorous TissueTable 3a -the Hong Kong Validation Cohort Fold change in microRNA ΔΔCt¹SD (ΔΔ Ct) tumors² p-value³ miR-20a 1.18 0.97 2.3 fold p < 0.001 miR-211.47 1.20 2.8 fold p < 0.001 miR-106a 1.25 0.94 2.4 fold p < 0.001miR-181 b 0.47 1.03 1.4 fold p < 0.001 miR-203 0.83 1.40 1.8 fold p <0.001 Table 3b MicroRNA Expression in Adenoma vs. Paired Non-adenomaTissue Average Fold change in microRNA ΔΔ Ct¹ SD (ΔΔ Ct) adenomas²p-value³ miR-20a −0.11 0.97 0.9 fold p = 0.82 miR-21 0.64 0.90 1.6 foldp = 0.006 miR-106a 0.28 1.22 1.2 fold p = 0.19 miR-181 b 0.30 1.24 1.2fold p = 0.27 miR-203 0.77 1.98 1.7 fold p = 0.14 ¹Average (tumor ΔCt −paired non-tumor ΔCt) or Average (adenoma ΔCt − paired nonadenoma ΔCt)from qRT-PCR. ²Calcluated by 2^(ΔΔ) ³Wilcoxon matched pairs test. SD =standard deviation. Bolded numbers are statistically significant. Forthe tumor/nontumor comparisons, 113 pairs of tissues were used formiR-20a and miR-203 while 111 pairs of tissue were used for miR-21,miR-106a, and miR-181b. For all adenoma/non-adenoma comparisons, 18pairs of tissue were used.

Most tumors (89% for miR-20a, 87% for miR-21, 90% for miR-106a, 71% formiR-181b and 74% for miR-203) had higher expression of these microRNAsthan paired nontumorous tissue. Expression patterns for these fivemicroRNAs distinguish tumor versus paired nontumor status with 96% or98% accuracy based on 3-nearest neighbors or nearest centroidalgorithms, respectively (10-fold cross validation, repeated 100 times).

We used in situ hybridization to visualize miR-21 expression in tumorand adjacent non-tumor tissue (see FIGS. 1 a-f).

MiR-21 is expressed at high levels in both the nuclei and cytoplasm ofcolonic epithelial cells in human tumor tissue compared to adjacentnontumorous tissue. These results are consistent with the qRT-PCR andmicroarray data and support a role for microRNAs in carcinogenesis.

MiR-21 is Expressed at Higher Levels in Colon Adenomas

Adenomas represent a precursor stage for colon adenocarcinomas. Wetested miR-20a, miR21, miR-106a, miR-181b and miR-203 expression levelsby qRT-PCR in 18 pairs of adenoma and adjacent nonadenoma tissue.Although four of five microRNAs showed increased levels in adenomatissue, only miR-21 was significantly enriched at 1.6-fold higher(p=0.006, Wilcoxon matched pairs test) (see Table 3b).

Adenoma tissue expressed higher levels of miR-21 in 15/18 matched pairs.More advanced stages of tumors express higher levels of miR-21. Subjectswere stratified based on the diagnosis of adenoma and TNM staging, whereadenoma was considered the least advanced and TNM Stage IV was mostadvanced. Adenomas expressed lower levels of miR-21 expression thantumors from the validation cohort (p<0.001, Mann-Whitney test). Moreadvanced tumors expressed higher levels of miR-21 expression (test fortrend, p<0.001) (see FIG. 1 g).

This trend was also observed using microRNA microarray data from theMaryland test cohort (p=0.04) (see FIG. 2).

High miR-21 Expression Predicts a Poor Prognosis in Two IndependentCohorts

We analyzed individual microRNA tumor/nontumor (T/N) expression ratiosto determine if any were associated with poor prognosis. T/N microRNAexpression ratios were classified as high based on highest tertile. Wesearched for any microRNA where high TIN ratios were associated withcancer survival (p<0.05). From those, we selected microRNAs that weredifferentially expressed in tumors (p<0.001). Five microRNAs satisfiedthese criteria. Kaplan-Meier analysis indicated that high T/N ratios formiR-20a (p=0.02), miR-21 (p=0.004), miR-106a (p=0.01), miR-181b(p=0.04), and miR-203 (p=0.004) were each associated with a poorsurvival. These five microRNAs were selected for further analysis.

Colon adenocarcinomas from 89-93% of the subjects in this study were ofa typical histology. A minority of tumors were of mucinousadenocarcinoma, adenosquamous carcinoma, or signet ring cell carcinomahistologies (see Table 1). Different subtypes of adenocarcinomas can beassociated with different clinical outcomes, including survivalprognosis. To remove potential confounding associated with histology, weexcluded all subjects with mucinous adenocarcinomas, adenosquamouscarcinomas and signet ring cell carcinomas from the initial analysis.

Associations of T/N ratios with poor survival could be due to microRNAexpression levels in the tumor tissue, the surrounding nontumoroustissue, or a combination of both. To distinguish these possibilities weanalyzed the association of microRNA expression in tumors and pairednontumors separately. High expression levels in tumors (based on highesttertile) for miR-20a, miR-21, miR106a, miR-181b and miR-203 were eachassociated with a poor survival in the Maryland test cohort (see FIG. 3a, also from data not shown). No significant association with microRNAexpression in nontumorous tissue was observed for any of the fivemicroRNAs.

Univariate and multivariate Cox proportional hazards analysis was usedto evaluate the association of tumor expression levels with prognosis inindividuals with typical adenocarcinoma (Table 4a).

TABLE 4 Univariate and Multivariate Cox Regression Analysis of miR-21Expression Levels and Overall Cancer Survival in Subjects with ColonAdenocarcinoma Table 4a Maryland Test Cohort Univariate analysisMultivariate analysis2 Characteristic HR (95% CI) p-value HR (95% CI)p-value miR-21 expression³ N = 71 Low 1.0 1.0 High 2.5 (1.2-5.2) 0.012.9 (1.4-6.1) 0.004 TNM Stage I-II 1.0 1.0 III-IV 3.5 (1.6-7.9) 0.0023.4 (1.5-7.8) 0.004 Age at enrollment <50 1.0 ≧50 0.7 (0.2-2.3) 0.52 SexFemale 1.0 Male 1.4 (0.5-3.9) 0.57 Race White 1.0 Black 1.0 (0.5-2.1)0.97 Tumor Location Distal 1.0 Proximal 0.6 (0.3-1.4) 0.26 Table 4b HongKong Validation Cohort Univariate analysis Multivariate analysis²Characteristic HR (95% CI) p-value HR (95% CI) p-value miR-21expression³ n = 103 Low 1.0 1.0 High 2.4 (1.4-3.9) 0.002 2.4 (1.4-4.1)0.002 TNM Stage I-II 1.0 1.0 III-IV 4.7 (2.4-9.5) <0.001 4.7 (2.4-9.5)<0.001 Age at enrollment <50 1.0 ≧50 1.5 (0.9-2.6) 0.14 Sex Female 1.0Male 1.4 (0.8-2.3) 0.29 Tumor Location Distal 1.0 Proximal 0.7 (0.3-1.4)0.27 MicroRNA expression was measured with miRNA microarrays for theMaryland cohort and with qRT-PCR with the Hong Kong cohort. ¹Cases withmucinous adenocarcinoma, adenosquamous carcinoma or signet ring cellcarcinomas were excluded from this analysis. ²Multivariate analysis usedstepwise addition and removal of clinical covariates found to beassociated with survival in univariate models (p < 0.10) and finalmodels include only those covariates which were significantly associatedwith survival (Wald statistic p < 0.05). ³High expression in tumors forall miRNAs was defined based on the highest tertile.

Individuals with tumors expressing high levels of miR-21 were at asignificantly higher risk of dying from colon cancer in both univariate(HR=2.5 [1.2-5.2], p=0.01) and multivariate (HR=2.9 [1.4-6.1], p=0.004)analyses.

To validate these findings, we used qRT-PCR to measure tumor andnontumor expression levels for these five microRNAs in the Hong Kongvalidation cohort and analyzed associations with prognosis. High miR-21tumor expression predicts a poor prognosis in the Hong Kong validationcohort (p=0.001, Kaplan-Meier log rank test) while expression innontumorous tissue does not (see FIG. 3 b).

We did not find statistically significant associations with prognosisand expression of miR-20a, miR-106a, 181b or miR-203 in this cohort.

High miR-21 expression in tumors was not significantly associated withage, gender, tumor histology, or tumor location (Fisher's exact test) inthe Hong Kong validation cohort. All covariates were examined by Coxproportional hazards analysis (Table 4b).

High miR-21 expression in tumors (HR⁼2.4 [1.4-3.9], p⁼0.002) and TNMstaging (HR=4.7 [2.4-9.5], p<0.001) were significantly associated withsurvival in univariate models. Multivariate Cox regression analysisdemonstrated that high miR-21 expression in tumors predicts poorsurvival prognosis (HR=2.4 [1.4-4.1], p=0.002) independent of otherclinical covariates, consistent with our findings in the Maryland testcohort.

We repeated the analysis including all subjects regardless of tumorhistology. In both cohorts, the association with high miR-21 expressionand prognosis remained (See FIG. 4, See Table 5).

TABLE 5 Univariate and Multivariate Cox Regression Analysis of miR-21Expression Levels and Overall Cancer Survival in Subjects with AllSubjects Table 5a Maryland Test Cohort Univariate analysis Multivariateanalysis Characteristic HR (95% CI) p-value HR (95% CI) p-value miR-21expression³ N = 79 Low 1.0 1.0 High 2.0 (1.1-4.0) 0.04 2.1 (1.1-4.0)0.03 TNM Stage I-II 1.0 1.0 III-IV 3.2 (1.5-6.9) 0.002 3.2 (1.5-6.8)0.003 Age at enrollment <50 1.0 ≧50 0.7 (0.2-2.4) 0.59 Sex Female 1.0Male 1.6 (0.7-4.2) 0.33 Race White 1.0 Black 1.0 (0.5-2.0) 0.99 TumorLocation Distal 1.0 Proximal 0.8 (0.3-2.1) 0.65 Histology 1.0Adenocarcinoma Mucinous or 0.7 (0.3-2.1) 0.57 Adenosquamous Table 5bHong Kong Validation Cohort Univariate analysis Multivariate analysis²Characteristic HR (95% CI) p-value HR (95% CI) p-value miR-21expression³ n = 111 Low 1.0 1.0 High 2.3 (1.4-3.9) 0.002 2.3 (1.4-3.9)0.002 TNM Stage I-II 1.0 1.0 III-IV 4.9 (2.5-97) <0.001 4.9 (2.5-98)<0.001 Age at enrollment <50 1.0 ≧50 1.4 (0.8-2.4) 0.20 Sex Female 1.0Male 1.3 (0.8-2.3) 0.27 Tumor Location Distal 1.0 Proximal 0.7 (0.3-1.4)0.27 Histology 1.0 Adenocarcinoma Mucinous or 1.2 (0.4-3.3) 0.74Adenosquamous MicroRNA expression was measured with miRNA microarraysfor the Maryland cohort and with qRT-PCR with the Hong Kong cohort. ¹Allindividuals were included in this analysis regardless of tumorhistology. ²Multivariate analysis used stepwise addition and removal ofclinical covariates found to be associated with survival in univariatemodels (p < 0.10) and final models include only those covariates whichwere significantly associated with survival (Wald statistic p < 0.05).³High expression in tumors for all miRNAs was defined based on thehighest tertile.

MiR-21 Expression Levels and Response to Therapy

Identifying biomarkers associated with a response to adjuvantchemotherapy will allow physicians to better predict the benefits oftherapy. To this end, we analyzed associations with miR-21 expressionand the response to adjuvant chemotherapy in stage II and III cancerpatients. Information on the administration of adjuvant chemotherapy wasavailable for 47 of 65 stage II or III subjects in the Maryland testcohort and all subjects in the Hong Kong validation cohort.

In both cohorts, chemotherapy regimens were primarily fluorouracil-based(in forms of either intravenous 5fluorouracil or oral drugs includingtegafur with uracil [UFT]) with or without Levamisole or Leucovorin.Only subjects with typical adenocarcinoma histology were used for thisanalysis, leaving 20 of 42 stage II/III individuals who receivedchemotherapy in the Maryland cohort. For those who receivedchemotherapy, high miR-21 expression in tumors predicted worse overallsurvival (p=0.01, Kaplan-Meier log rank test) giving preliminary supportthat high miR-21 is associated with poor response to adjuvantchemotherapy.

For the Hong Kong validation cohort, 77 individuals with stage II/IIIcancer with typical adenocarcinoma histology were used for thisanalysis. Stage II/III subjects who received adjuvant chemotherapy hadbetter survival prognosis than those who did not (p=0.02, Kaplan-Meierlog rank test). Among those subjects that received adjuvant chemotherapy(n=36), high miR-21 expression in tumors was associated with a poorresponse to treatment (p=0.03, Kaplan-Meier log rank test), consistentwith observations in the Maryland cohort (see FIG. 5 a).

In this cohort, all stage II subjects who received adjuvant chemotherapy(n=11) survived (see FIG. 5 b), but for stage III subjects who receivedadjuvant chemotherapy (n=25), high miR-21 expression was associated withpoor survival (p=0.02, Kaplan-Meier log rank test) (see FIG. 5 c).

Multivariate Cox regression analysis was used to analyze theseobservations to show that high miR-21 expression predicted a poorprognosis (HR=3.1 [1.5-6.1]; p=0.001) and receiving chemotherapy waspredictive of improved survival outcomes (HR=0.3 [0.1-0.5]; p<0.001)independent of other clinical covariates (Table 6a).

TABLE 6 Univariate and Multivariate Cox Regression Analysis of miR-21Expression, Receipt of Adjuvant Chemotherapy and Cancer Survival inStage I/III¹ Subjects with Adenocarcinoma Table 6a Maryland Test CohortUnivariate analysis Multivariate analysis² Characteristic HR (95% CI)p-value HR (95% CI) p-value miR-21 expression³ N = 77 Low 1.0 1.0 High2.6 (1.3-5.1) 0.005 3.1 (1.5-6.1) 0.001 Adjuvant Chemotherapy Did notreceive 1.0 1.0 Received 04. (0.2-0.8) 0.01 0.3 (0.1-0.5) <0.001 TNMStage II 1.0 1.0 III 2.8 (1.3-6.0) 0.008 5.4 (2.4-12)  <0.001 TumorLocation Distal 1.0 1.0 Proximal 0.3 (0.1-1.0) 0.04 0.2 (0.1-0.8) 0.02Age at enrollment <50 1.0 ≧50 1.6 (0.8-3.1) 0.20 Sex Female 1.0 Male 1.2(0.6-2.3) 0.61 Table 6b Hong Kong Validation Cohort Univariate analysisMultivariate analysis² Characteristic HR (95% CI) p-value HR (95% CI)p-value miR-21 expression³ N = 119 Low 1.0 1.0 High 2.6 (1.5-4.5) 0.0013.0 (1.7-5.4) <0.001 Adjuvant Chemotherapy Did not receive 1.0 1.0Received 07. (0.4-1.2) 0.21 0.4 (0.2-0.8) 0.004 TNM Stage II 1.0 1.0 III3.2 (1.7-6.1) 0.001 5.2 (2.6-11)  <0.001 Tumor Location Distal 1.0 1.0Proximal 0.4 (0.2-0.8) 0.02 0.3 (0.1-0.7) 0.007 Age at enrollment <501.0 ≧50 1.4 (0.7-2.5) 0.32 Sex Female 1.0 Male 1.3 (0.7-2.2) 0.44Expression of miRNAs was measured with qRT-PCR. ¹TNM stage II/IIIsubjects with typical adenocarcinoma histology were included in thisanalysis. ²Multivariate analysis used stepwise addition and removal ofclinical covariates found to be associated with survival in univariatemodels (p < 0.10) and final models include only those covariates whichwere significantly associated with survival (Wald statistic p < 0.05).³High expression in tumors for all miRNAs was defined based on thehighest tertile. Race was not associated with poor prognosis.

Analyses using cancer relapse as an endpoint instead of cancer deathresulted in similar associations with high miR-21 expression in tumorspredicting a more rapid disease recurrence (data not shown).

An analysis combining both cohorts resulted in similar associations.Kaplan-Meier analysis demonstrated that high miR-21 expression predicteda poor prognosis in either stage II (p=0.02) or stage III (p=0.004)subjects (See FIG. 6).

High miR-21 expression predicted a poor response to chemotherapy instage II/III subjects (p=0.003) or in stage III subjects alone(p=0.007). Multivariate Cox regression demonstrated that high miR-21expression predicted poor prognosis (HR=3.0 [1.7-5.4]; p<0.001) andtreatment with adjuvant chemotherapy predicted improved survival (HR=0.4[0.2-0.8]; p=0.004) independent of other clinical covariates (Table 6b).

Discussion

We analyzed microRNA profiles in colon cancer tissues using twoindependent cohorts. Thirty-seven microRNAs were differentiallyexpressed in tumor tissues by microRNA microarray analysis. Expressionpatterns of all five tested microRNAs were validated in the Hong Kongcohort. The discriminatory power of five microRNAs to differentiatebetween tumor and nontumorous tissue indicates that predictable andsystematic changes of microRNA expression patterns occur duringtumorigenesis and are likely representative of the majority of sporadiccolon adenocarcinomas.

MiR-20a, miR-21, miR-106a, miR-181b and miR-203 were all found to beexpressed at higher levels in colon tumors. These changes in microRNAexpression patterns may be merely associated with colon cancer or causalto the histologic progression to cancer. There is strong evidencesuggesting that changes in microRNA expression patterns promote tumorformation, especially for miR-20a and miR-21. MiR-20a is part of themiR-17-92 polycistronic microRNA cluster.

Overexpression of this cluster enhances cell proliferation in vitro andaccelerates tumor formation in animal models. Enforced expression of themiR-17-92 cluster causes increased tumor size and tumor vascularizationin mice by negatively regulating the anti-angiogenic Tspl protein.Experimental evidence also suggests that increased miR-21 expressionpromotes tumor development. MiR-21 is expressed at high levels in mostsolid tumors. Overexpression of miR-21 acts as an anti-apoptotic factorin human glioblastoma cells Inhibition of miR-21 inhibits cell growth invitro and inhibits tumor growth in xenograft mouse models through anindirect downregulation of the anti-apoptotic factor Bcl-2. Studies inhuman cell lines have shown miR-21 can also target the tumor suppressorgenes PTEN³ and TPM1. All of these data taken together support a causalrole for altered microRNA expression during tumorigenesis.

Adenomas represent a precursor stage of adenocarcinoma. Adenomas expresshigh levels of miR-21. If increased miR-21 expression promotes colontumor progression, increased expression in adenomas may be an earlycellular event in the progression to cancer. Inhibiting miR-21 activitymay help prevent tumor promotion in populations at high risk for coloncancer, such as individuals with familial adenomatous polyposis.

Thus, there is presented herein evidence that demonstrates anassociation with microRNA expression patterns with colon cancerprognosis and response to adjuvant chemotherapy. More advanced tumorsexpress higher levels of miR-21. A robust association with high miR-21expression in tumors and poor survival was observed in the Maryland testcohort and the Hong Kong validation cohort, separately.

In each cohort, these associations were independent of all otherclinical covariates indicating that miR-21 expression may be a usefulprognostic indicator, in addition to TNM staging and other clinicalparameters, to help identify patients at a higher risk of terminalcancer. These observations were made in two independent cohorts withvery different racial and geographical compositions. Therefore, it islikely that our observations are broadly applicable to otherpopulations.

High miR-21 expression in tumors was associated with a poor response toadjuvant chemotherapy in both cohorts. These results can help predictthe benefits of therapy in individuals whose miR-21 expression status isknown. In addition, if high miR-21 expression is causal to the poorsurvival of colon cancer patients, antagomirs or other antisensetherapeutics that target miR-21 can have therapeutic benefits insubjects with high miR-21 expressing tumors. These may be used inaddition to current therapies to improve survival outcomes.

The inventors herein have found systematic differences in microRNAexpression patterns between colon tumors and paired nontumorous tissue.High miR-21 expression in tumors predicts poor survival outcome and poorresponse to adjuvant chemotherapy in two independent cohorts,independent of staging and other clinical covariates suggesting that itmay be a useful diagnostic biomarker for colon adenocarcinomas andsurvival prognosis including response to therapy.

Methods

Tissue Collection and RNA Isolation:

Pairs of primary colon tumor and adjacent nontumorous tissues came from84 patients recruited from the University of Maryland Medical Centerbetween 1993 and 2002, and from 113 patients recruited from Queen MaryHospital in Hong Kong between 1991 and 2000. Detailed backgrounds foreach tissue donor, including age, gender, clinical staging, tumorlocation, survival times from diagnosis and receipt of adjuvantchemotherapy have been collected. Tumor histopathology was classifiedaccording to the World Health Organization Classification of Tumorsystem¹. The adenoma tissue was obtained from the Cooperative HumanTissue Network. This study was approved by the Institutional ReviewBoard of the National Institutes of Health, the Institutional ReviewBoard of the University of Hong Kong/Hospital Authority Hong Kong WestCluster and the Institutional Review Board for Human Subject Research atthe University of Maryland.

RNA Isolation and microRNA Profiling:

RNA was extracted from tissue using standard TRIZOL (Invitrogen,Carlsbad) methods. MicroRNA microarray profiling was performed aspreviously described. Briefly, 5 lag of total RNA was labeled andhybridized to each microRNA microarray containing quadruplicates ofapproximately 400 human microRNA probes. Slides were scanned using aPerkinElmer ScanArray LX5K scanner. qRT-PCR of microRNAs was performedusing Taqman MicroRNA assays (Applied Biosystems, Foster City) accordingto manufacturer's instructions with the 7500 real time RT-PCR system(Applied Biosystems, Foster City). U6B was the normalization control forall qRT-PCR experiments. All assays were performed in duplicate(miR-20a, miR-203) or triplicate (miR-21, miR-106a, miR-181b). qRT-PCRfor miR-21, miR-106a and miR-181b was performed by AJS, who was blindedto the survival outcomes and clinical data for members of the validationcohort at that time.

Microarray Analysis:

The data discussed in this publication have been deposited in NCBIs GeneExpression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and areaccessible through GEO Series accession number GSE7828. LOESS normalizedmicroarray data were imported into BRB array tools 3.5.0(http://linus.nci.nih.gov/.BRB-ArrayTools.html) and all subsequentmicroarray analyses were performed with this software.

Microarray analyses were performed. Probes with values missing from >20%of the arrays were removed from the analysis leaving 230 probes. Paired,class comparison analysis identified microRNAs that were differentiallyexpressed in tumors (p<0.001).

To initially search for microRNAs associated with poor survival,tumor/nontumor (T/N) microRNA expression ratios were analyzed in theMaryland cohort using microarray data. TN expression ratios formicroRNAs were created by subtracting the log₂ nontumor from the log₂tumor expression values. MicroRNAs missing >25% of T/N ratios werefiltered out leaving 208. T/N expression ratios were dichotomized withthe highest tertile classified as high and the lower 2 tertilesclassified as low (see Supplemental Methods). This high/low cutoff wasused universally throughout this study. Tumor and nontumor microRNAexpression levels were batch normalized based on the date of microarrayexperiments for all analysis of associations with survival.

In Situ Hybridization:

In situ hybridization (ISH) was performed with probes for human miR-21,scramble, and U6 (Exiqon, Woburn) with a modified version of themanufacturer's protocol for formalin-fixed paraffin-embedded (FFPE)tissue written by W. Kloosterman (http://www.exiqon.com/uploads/LNA52-FFPE miRNA in situj,rotocol.pdf) on human colon tissue. Modificationsincluded the use of polyclonal rabbit anti-DIG/HRP-conjugated antibodyand DakoCytomation GenPoint Tyramide Signal Amplication System(DakoCytomation, Carpinteria), and VECTOR® NovaRed™ substrate (VectorLaboratories, Burlingame). Images were taken on an Olympus BX40microscope using the Olympus DP70 digital camera and DP controllersoftware (Olympus, Champaign).

Statistical Analysis:

Statistical analyses were performed. Wilcoxon matched pairs tests wereused to analyze differences in microRNA expression between tumors andpaired nontumorous tissue as well as differences between adenoma andpaired non-adenoma tissue for all qRT-PCR data. All trend tests reportedare nonparametric tests for trend across ordered groups. AllKaplan-Meier analysis was performed with WINSTAT 2001 (R. FitchSoftware). Multivariate Cox regression analysis was performed usingIntercooled Stata 9.2 (StataCorp LP, College Station). Finalmultivariate models were based on stepwise addition and removal ofclinical covariates found to be associated with poor survival inunivariate models (p<0.10). A Wald statistic of p<0.05 was used ascriteria for inclusion in final multivariate models. All p-valuesreported are 2-sided. Hazards ratios are reported with 95% confidenceintervals in parentheses. Expression graphs were made using GraphpadPrism 4.0 (Graphpad Software Inc., San Diego).

Additional Microarray Analyses

The microarrays used for this analysis were pin-spotted microRNAmicroarrays (from the Ohio State University Comprehensive Cancer Center,version 2.0). Intensities of each spot were the median intensities offoreground. Each of the 170 microarrays used for this study contained11520 spots. All spots where foreground intensity was less thanbackground were reassigned as NA (NA marks missing data spots). Allspots flagged as deficient by the scanner were also reassigned as NA.All blank (no oligo) spots with high foreground intensity werereassigned as NA. Each microRNA oligo is represented by quadruplicatespots on these arrays as two distant pairs of two adjacent spots. Ifthere were 0 or 1 NA for an oligo quadruple, and the means of thedistant oligo pairs differed by >1 on the log₂ scale, all of thequadruplicate spots were reassigned as NA. If there were 2 NAs for anoligo quadruple and the two non-NA spot intensities differed by >1 onthe log₂ scale, all of the quadruplicate spots were reassigned NA. Ifthere were 3 NA spots for a quadruple, the final spot was reassigned asNA. In total, 1,082,689 of 1,958,400 spots were reassigned as NA usingthese methods. LOESS (Locally Weighted Scatterplot Smoothing)normalization was performed using the R software package. All data wasthen imported into BRB array tools version 3.5.0 for analysis and allreplicate spots were averaged. There were originally 85 pairs (tumor andpaired nontumorous tissue) of arrays used. One case that was originallyidentified as an incident colon carcinoma patient was later found tohave been diagnosed as carcinoma in situ and was removed from theanalysis leaving the study population of 84 subjects. MicroRNA listswere filtered to include only the 389 human hsa-miR probesets. They werefurther filtered to remove any probeset missing from more than 25% ofthe arrays, leaving 230 human microRNA probesets. Paired classcomparison analysis was used to identify microRNAs that weredifferentially expressed between tumor and paired nontumorous tissue.For two microRNAs (miR-181b and miR-338), two independent probesmeasuring each gave contradictory results with one probe showing higherexpression in tumors and one probe showing lower expression in tumorsfor each microRNA. For each, we discarded the less significant resultwhich designated both miR-181b and miR-388 as enriched in tumors.Additionally, qRT-PCR confirmed that miR-181b was enriched in tumors.

We initially used tumor/nontumor (T/N) expression profiles for eachmicroRNA to search for microRNAs that were associated with poorsurvival. For this analysis, we decided to dichotomize all expressiondata with a universal high and low cutoff to look for associations withpoor survival. To determine what universal high/low cutoff to use, wedichotomized the T/N expression data three separate ways and determinedwhich method gave the greatest number of significant results in the testcohort. High expression was classified based on higher than median,highest tertile, or highest quartile and we tested associations withthese cutoffs with a poor survival using univariate Cox regressionanalysis. Of the 37 microRNAs that were differentially expressed intumors, high expression of four were associated with poor survival basedon higher than median, five based on highest tertile, and two based onhighest quartile (p<0.05, data not shown). Dichotomization based onhighest tertile gave the most microRNAs associated with poor survivalbased on these criteria in the Maryland test cohort; therefore,classification based on highest tertile was used uniformly throughoutthis study to analyze associations between microRNA expression levelsand a poor prognosis in both the Maryland test cohort and the Hong Kongvalidation cohort.

We used microRNA microarrays to compare miR-21 expression levels intumors with prognosis. The microarray probe used for this analysis washsa-miR-21-prec17No1. This analysis required batch normalization of thedata based on the date of the microarray experiment. To normalize bydate, arrays expressing the highest ⅓ of a given microRNA wereclassified as high for each day, separately. Up to twelve pairs oftissue were profiled on any given day. For any day in which less than 10pairs of microarrays were performed, arrays performed on those days werediscarded, resulting in the loss of 5 pairs of arrays. These data werethen combined together for analysis of associations with survivaloutcomes. We checked and found no significant differences in thefrequency distribution of age, sex, race, tumor location, TNM stage, orcancer survival between groups categorized based on date of microarrayexperiment (Fisher's exact test).

Statistical Analyses

Cox proportional hazards regression was used to analyze the effect ofmir-21 expression levels and other clinical variable on patientsurvival. Clinical variables included were age, sex, race, tumorlocation, tumor histology, receipt of adjuvant therapy and TNM staging.For these models, we chose to dichotomized age as age >50 versus age <50as the recommended screening age for colon cancer is at age 50; tumorlocation was defined as proximal if tumor was located within or proximalto the splenic flexure and distal if tumor was located within or distalto the descending colon; TNM staging was dichotomized based onmetastasic versus nonmetastasic disease resulting in stage I-II versusIII-IV. One patient in the Maryland cohort died on the day of surgeryresulting in a survival time of 0 months. This case was included inKaplan-Meier analysis and removed for Cox regression analysis causingthe difference in cases between miR-21 expression in tumors for FIG. 2(n=72) and the number of cases in the Table 4 Cox regression analysis(n=71). Univariate Cox regression was performed on each clinicalcovariate to examine influence of each on patient survival. Finalmultivariate models were based on stepwise addition and removal ofclinical covariates found to be associated with poor survival inunivariate models (p<0.10). A Wald statistic of p<0.05 was used ascriteria for inclusion in final multivariate models. The mostparsimonious Cox regression model was used for the final multivariatemodel.

Example 2 Initial Results

MiRNAs are Differentially Expressed in Colon Tumors

We analyzed miRNA profiles of 85 pairs of cancerous and adjacentnon-cancerous colon tissues using miRNA microarrays. We found that miRNAexpression profiles of tumors were quite different than normal tissuessuggesting that miRNAs may play significant roles in coloncarcinogenesis. Paired class comparison analysis identified 27independent miRNAs that were differently expressed in these tumors(Table 7).

TABLE 7 Table 7 - 27 miRNAs are differentially expressed in colon tumorscompared to paired, normal tissue. 27 miRNAs were found to bedifferentially expressed in tumors using paired class comparisonsanalysis in BRB array tools 3.4. A significance value of p < 0.001 wasused as the criteria for differentially expressed which resulted in anestimated false discovery rate of 0.08%. Up refers to miRNAs that wereexpressed at higher levels in tumors while down indicates that miRNAlevels were lower in tumors. MicroRNA Up/Downregulated P-Value 1 miR-331Down 1.00E−07 2 miR-21 Up 1.00E−07 3 miR-34b Down 2.00E−07 4 miR-342Down 2.00E−07 5 miR-215 Down 2.20E−05 6 miR-371 Down 7.00E−07 7 miR-373Down 6.30E−06 8 miR-192 Down 7.70E−06 9 miR-148b Down 1.03E−05 10miR-138 Down 1.49E−05 11 miR-301 Down 1.85E−05 12 miR-338 Down 2.63E−0513 miR-153 Down 2.67E−05 14 miR-129 Down 3.20E−05 15 miR-222 Up 9.08E−0516 miR-346 Up 0.000126 17 miR-204 Up 0.000244 18 miR-181 b Up 0.00026319 let-7a-2 Down 0.000272 20 miR-106a Up 0.000305 21 miR-093 Up 0.00033422 miR-34c Down 0.000341 23 miR-219 Up 0.000352 24 miR-019b Up 0.00036425 miR-210 Up 0.000389 26 miR-185 Up 0.000516 27 miR-1 Down 0.00064

The false discovery rate, to account for the multiple comparisonstesting, was approximately 0.8% indicating that most, if not all ofthese miRNAs are differentially expressed and not the result of multiplecomparisons testing. Eleven miRNAs were found to have elevatedexpression levels in tumors while 16 miRNAs were found to be reduced intumors. Additionally, miRNA profiles could be used to predict whether ornot the tissue was tumor or non-tumor with 92% accuracy. Based on 2000random permutations, the probability of these predictions occurring byrandom chance was extremely low (p<0.0005). These results show thatthere are systematic differences in miRNA expression profiles betweentumors and normal tissue indicating that miRNA expression profilesbecome altered during colon carcinogenesis.

Global miRNA Expression Profiles Predict Colon Cancer Survival Prognosis

We determined whether miRNA expression profiles predict patientsurvival. For this analysis we calculated the tumor versus normal miRNAexpression ratios (TIN ratio) for each miRNA for every individual.Unsupervised hierarchical clustering of all miRNA TIN ratios groupedindividuals into two groups arbitrarily labeled group A and group B(FIG. 7).

These two groups differ significantly in both clinical staging (p=0.009;FIG. 1 b) and survival prognosis (p=0.026; FIG. 7 c).

This indicated that global miRNA profiles are predictive of clinicalstaging and more importantly, survival prognosis.

Univariate and multivariate Cox regression analysis was used tointerrogate this relationship in more detail (Table 8).

TABLE 8 Cox Regression Analysis of global miRNA Profiles Univariate(above) and multivariate (below) Cox regression analyses were performedto show that individuals classified in miRNA group B were at higher riskof dying of colon cancer. Neither age, gender or race was significantcontributors to survival risk. For the purposes of these analyses, agewas dichotomized into greater than or less than 50 and race dichotomizedinto African American (AA) and Caucasian. Variable HR (95% Cl) p valueUnivariate Analysis Cluster B/A 2.6 (1.0-6.3) 0.042 age ≧ 50/age < 500.62 (0.14-−2.7) 0.53 male/female 1.4 (0.48-4.0) 0.54 AA/Caucasian 1.1(0.83-2.3) 0.83 Multivariate, adjusting for age, gender and race ClusterB/A 2.7 (1.1-6.8) 0.034 age ≧ 50/age < 50 0.49 (0.11-−2.2) 0.35male/female 1.5 (0.52-4.4) 0.45 AA/Caucasian 1.0 (0.45-2.2) 0.99

Group B individuals were to have a significantly higher risk of dyingfrom colon cancer (hazard ratio [HR]=2.6 (p=0.04). This risk remainedsignificantly high after adjusting for age, ethnicity and gender(HR=2.7; p=0.03). These results demonstrate the potential for usingmiRNA profiles of colon tumors to predict prognosis. These resultssuggest that miRNAs may also play a role in colon carcinogenesis.

Profiles of miR-21, miR-106a, miR-181b, miR-16b, miR-203, let-7g,miR-29a, miR-103-2 and miR-10a Predict Colon Cancer Prognosis

We identified individual miRNAs whose expression levels were predictiveof colon cancer prognosis. We used Kaplan Meier survival plots andmultivariate Cox regression analysis on TIN ratios to identify miRNAexpression patterns that were associated with poor survival prognosis.BRB array tools were used to identify TIN ratios correlated with poorsurvival (data not shown). We chose to analyze these miRNAs in furtherdetail. We also analyzed any miRNA that was differentially expressed intumors (p<0.01). TIN ratios for each individual were dichotomized basedon median or highest quartile TIN ratios. We also removed any miRNA fromthe analysis where TIN ratios were missing in greater than 18individuals. We identified at least 9 miRNAs, including miR-21,miR-106a, miR-181b, miR-16h, miR-203, let-7g, miR-29a, miR-103-2 andmiR-10a whose TIN ratios are predictive of colon cancer prognosis (FIG.8, Table 9).

Cox Regression Analysis of TIN Ratios for Individual miRNAs.

Univariate and multivariate Cox regression analyses were performed toshow that TIN ratios of individual miRNAs could by used to classifyindividuals at higher risk of dying of colon cancer. TIN ratios forthese 9 miRNAs were significant predictors of survival prognosisindependent of TNM staging, age, gender and race. Note that High/Lowdistinctions for miR-16b, miR-21, miR-29a, miR-103-2, miR-106a andmiR-203 were classified based on median TIN ratio values while let-7g,miR-10a and miR-181b were classified based on highest quartile TINratios.

TABLE 9 Cox regression analysis of TIN ratios for individual miRNAsVariable HR (95% Cl) p = n Univariate analysis miR-21 High/Low 3.0(11.3-7.0) 0.01 80 Multivariate analysis miR-21 High/Low 2.8 (1.2-6.8)0.02 age ≧ 50/age < 50 0.46 (0.10-2.1) 0.32 male/female 3.1 (0.9-11.0)0.07 AA/Caucasian 1.2 (0.5-2.7) 0.66 Stage III-IV/Stage I-II 4.4(1.6-11.9) 0.004 Univariate analysis miR-181b High/Low 3.4 (1.6-7.5)0.002 78 Multivariate analysis miR-181b High/Low 3.3 (1.3-8.2) 0.01 age≧ 50/age < 50 0.39 (0.08-1.8) 0.23 Male/female 2.2 (0.7-7.2) 0.17AA/Caucasian 1.1 (0.5-2.5) 0.82 Stage III-IV/Stage I-II 3.1 (1.2-8.1)0.02 Univariate analysis let-7g High/Low 2.7 (1.3-5.9) 0.01 84Multivariate analysis let-7g High/Low 2.5 (1.1-5.5) 0.03 age ≧ 50/age <50 0.5 (0.1-2.4) 0.39 Male/female 1.5 (0.5-4.4) 0.50 AA/Caucasian 1.3(0.6-2.9) 0.50 Stage III-VI/Stage I-II 3.6 (1.4-9.2) 0.006. Univariateanalysis miR-103-2 High/Low 2.5 (1.1-5.6) 0.03 81 Multivariate analysismiR-103-2 High/Low 3.1 (1.3-7.5) 0.01 age ≧ 50/age < 50 0.5 (0.1-2.2)0.36 male/female 1.6 (0.6-4.9) 0.38 AA/Caucasian 0.8 (0.4-1.9) 0.69Stage III-IV/Stage I-II 4.4 (1.7-11.1) 0.002 Univariate analysis miR-16bHigh/Low 4.6 (1.7-12.5) 0.003 69 Multivariate analysis miR-16b High/Low5.1 (1.8-15.9) 0.003 age ≧ 50/age < 50 0.4 (0.08-1.7) 0.20 male/female3.2 (0.8-1.7) 0.12 AA/Caucasian 0.9 (1.9-22.4) 0.003 Stage III-IV/StageI-II 6.5 (1.9-22.4 0.003 Univariate analysis miR-106a High/Low 2.6(1.1-6.1) 0.01 82 Multivariate analysis miR-106a High/Low 2.4 (1.0-5.7)0.05 age ≧ 50/age < 50 0.54 (0.11-−2.5) 0.44 male/female 1.8 (0.5-6.5)0.34 AA/Caucasian 1.1 (0.5-2.5) 0.84 Stage III-IV/Stage I-II 5.4(1.8-16.0) 0.002 Univariate analysis miR-203 High/Low 3.8 (1.4-10.5)0.01 57 Multivariate analysis miR-203 High/Low 3.2 (1.1-9.4) 0.03 age ≧50/age < 50 1.0 (0.1-−8.1) 0.97 male/female 1.4 (0.4-5.1) 0.61AA/Caucasian 0.9 (0.4-2.3) 0.83 Stage III-IV/Stage I-II 3.9 (1.3-11.8)0.02 Univariate analysis miR-29a High/Low 3.1 (1.3-7.3) 0.01 77Multivariate analysis miR-29a High/Low 3.2 (1.3-7.9) 0.01 age ≧ 50/age <50 0.5 (0.1-−2.2) 0.35 male/female 2.2 (0.6-7.4) 0.22 AA/Caucasian 0.9(0.4-2.1) 0.76 Stage III-IV/Stage I-II 4.5 (1.7-12.2) 0.003 Univariateanalysis miR-10a High/Low 2.7 (1.3-5.7) 0.01 84 Multivariate analysismiR-10a High/Low 3.5 (1.5-7.8) 0.003 age ≧ 50/age < 50 0.4 (0.1-−1.9)0.26 male/female 1.7 (0.6-5.0) 0.34 AA/Caucasian 1.0 (0.45-2.3) 0.98Stage III-IV/Stage I-II 4.9 (1.9-12.2) 0.001

MiR-21 expression is elevated in tumors (Table 7). The miR-21 TIN ratiosare also associated with clinical staging and survival prognosis forcolon cancer patients as well (Table 9, FIG. 8 a).

There was a trend that individuals with more advanced TNM staging havehigher TIN ratios (p=0.034). TIN ratios were dichotomized based onmedian values for each of the 80 individuals with data. Individuals withhigh miR-21 TIN expression ratios had a worse survival prognosis basedon Kaplan Meier analysis (p=0.004) suggesting that tumors expressinghigh levels of miR-21 is predictive of poor prognosis. These resultswere further analyzed with Cox regression analysis.

Individuals with high TIN ratios of miR-21 were at higher risk with bothunivariate (HR=3.0; p=0.01) and multivariate (HR=2.8; p=0.02) analysisadjusting for age, gender, race and TNM staging (Table 9).

This result suggested that miR-21 expression levels can be useful asprognostic prediction methods and can provide more predictive value forsurvival prognosis than TNM staging alone. miR-21 has been found to bedifferentially expressed in many tumor types

Studies have also demonstrated that high levels of miR-21 can lead to aninhibition of apoptosis in glioblastoma cells while inhibition of miR-21can lead to increased cell proliferation in HeLa cells.

The inventors herein discovered that miR-21 is now believed to becontributing to colon carcinogenesis in a similar manner.

We found that miR-106a elevated in tumors (Table 7) and miR-106a TINratios are associated with survival prognosis (Table 9, FIG. 8 b).

MiR-106a is a member of a class of paralogous miRNAs including miR-17,miR-20, miR-106a, and miR-106h. These miRNAs are very similar to oneanother in that they differ by only 1-2 nucleotides. Due to theirsimilarity, they are all likely to have similar targets. Interestingly,all four of these miRNAs show similar patterns of expression andassociations with prognosis (data not shown). We present hereinassociations for miR-106a, but we do not formally rule out thepossibility that any or all of the other miRNA paralogs are contributingto this association. MiR-106a TIN ratios were dichotomized based onmedian values for each of the 82 individuals with data. Individuals withhigh miR-106a TIN expression ratios had a worse survival prognosis basedon Kaplan Meier analysis (p=0.013; FIG. 8 b).

This suggests that tumors expressing high levels of miR-106a arepredictive of poor survival prognosis. Individuals with high TIN ratiosof miR-106a were at higher risk with both univariate (FIR=2.6, p=0.01)and multivariate (HR=2.4; p=0.05) analysis adjusting for age, gender,race and TNM staging (Table 7). Therefore, miR-106a may be a usefulprognostic predictor of colon cancer prognosis independent of TNMstaging, Interestingly, the Retinoblastoma tumor suppressor gene hasbeen shown to be a functional target of miR-106a, supporting a mechanismof how miR-106a may be mechanistically contributing to coloncarcinogenesis.

Overexpression of the miR-17-92 cluster, which contains paralogs ofmiR-106a, resulted in accelerated tumor development in mice. Thisexperimentally shows that miRNAs of the miR-106a family are capable ofaffecting carcinogenesis further strengthening the hypothesis thatmiR-106a may be contributing to carcinogenesis and tumor progression.

Expression patterns of seven additional miRNAs were associated withclinical staging and poor survival prognosis (Table 9, FIGS. 8 c-8 i).

There is a trend that individuals diagnosed with more advanced TNMstaging had higher TIN ratios for let-7g (p=0.010), miR-10a (p=0.008),miR-16h (p=0.048), miR-29a (p=0.005), miR-103-2 (p=0.033), miR-181b(p=0.016), and miR-203 (p=0.016) (FIG. 8).

TIN ratios were dichotomized based on median (miR-16h, miR-29a,miR-103-2, miR-203) or highest quartile (let-7g, miR-10a, miR-181b) andKaplan Meier analysis revealed that high TIN ratios for each were foundto be predictors of poor survival prognosis (FIG. 8 c-8 i).

Univariate and multivariate Cox regression analysis confirmed that highTIN ratios of any one of these miRNAs were predictive of poor coloncancer prognosis independent of TNM staging (Table 9). Multivariate Coxregression models that adjusted for age, gender, race and TNM stagingshowed that high TIN ratios for miR-16b (HR=5.1; p=0.003), let-7g(HR=2.5; p=0.03), miR-10a (HR=3.4; p=0.003), miR-29a (HR=3.2; p=0.01),miR-103-2 (HR=3.1; p=0.01), miR-181b (HR=3.2; p=0.01), and miR-203(HR=3.2; p=0.03) were each predictive of poor survival prognosis. Theseresults suggested that patients with tumors expressing high levels ofany of these miRNAs are at an increased risk of dying from colon cancer.Therefore, expression levels of any these miRNAs may be usefulbiomarkers that can help predict survival risks for colon cancerpatients independent of staging.

MiRNA Expression Signature of 9 miRNAs Predicts Survival Prognosis:

We used the TIN ratios for all 9 of the previously mentioned miRNAs todevelop a miRNA signature that could be used to predict colon cancerprognosis. Individuals missing more than 2 of 9 of these values wereexcluded from this analysis. Hierarchical clustering of the TIN ratiosof the 9 miRNAs resulted in grouping the remaining 78 patients into twogroups (FIG. 9 a).

These groups had significantly different survival prognoses (FIG. 9 b;p=0.004). Univariate (HR=3.2, p=0.008) and multivariate (HR=2.8; p=0.04)Cox regression analysis demonstrated that the miRNA signature wasassociated with poor survival prognosis independent of TNM staging(Table 10)

TABLE 10 Cox regression analysis of microRNA signature Variable HR (95%Cl) p value Univariate Analysis 9 miR Cluster B/A 3.2 (1.4-7.8) 0.008Multivariate, adjusting for age, gender and race 9 miR Cluster B/A 2.8(1.0-7.4) 0.043 age ≧ 50/age < 50 0.4 (0.08-−1.8) 0.23 male/female 1.9(0.6-6.6) 0.29 AA/Caucasian 0.9 (1.4-10.7) 0.82 Stage III-IV/Stage I-II3.9 (1.4-10.7) 0.007

Univariate (above) and multivariate (adjusting for age, gender race andstaging; below) Cox regression analyses were performed to show thatindividuals classified into group B using the 9 miRNA signature were athigher risk of dying of colon cancer. Neither age, gender nor racesignificantly contributed to survival risk. This risk associated withcluster assignment is independent of staging.

These results demonstrate that miRNA signatures may be used as abiomarker to predict the survival prognosis of colon cancer patients.

Discussion

Individual miRNAs are differentially expressed in colon tumorssuggesting that altered expression of these miRNAs may be part of thecellular changes responsible for colon carcinogenesis. In addition tothese findings, we show herein that miRNA expression profiles areassociated with colon cancer staging and prognosis. Therefore miRNAs,either analyzed individually or as part of a miRNA signature, can beused as biomarkers that will enable physicians to predict patientsurvival risk with more accuracy.

The strong associations with miRNA TIN ratios with survival prognosissuggest that altered miRNA expression may be part of the causal pathwayin colon carcinogenesis and progression. If altered expression of any ofthese miRNAs is causal to carcinogenesis, it may be possible to designantagomir-like pharmaceuticals that can be used to treat cancer. UsingmiRNA profiling and miRNA based therapeutics, it may be possible todesign personalized drug treatment strategies based on which of thesenine miRNAs are altered. Additionally, these strategies may be useful inpreventing colon cancer in people that are at high risk due togenetically inherited risks or previous cancer history.

Example 3 Methods, Reagents and Kits for Diagnosing, Staging,Prognosing, Monitoring and Treating Colon Cancer-Related Diseases

In one embodiment, there is provided a diagnostic method of assessingwhether a patient has a colon cancer-related disease or has higher thannormal risk for developing a colon cancer-related disease, comprisingthe steps of comparing the level of expression of a marker in a patientsample and the normal level of expression of the marker in a control,e.g., a sample from a patient without a colon cancer-related disease. Asignificantly higher level of expression of the marker in the patientsample as compared to the normal level is an indication that the patientis afflicted with a colon cancer-related disease or has higher thannormal risk for developing a colon cancer-related disease.

The markers are selected such that the positive predictive value of themethods is at least about 10%, and in certain non-limiting embodiments,about 25%, about 50% or about 90%. Also preferred for use in the methodsare markers that are differentially expressed, as compared to normalcells, by at least two-fold in at least about 20%, and in certainnon-limiting embodiments, about 50% or about 75%.

In one diagnostic method of assessing whether a patient is afflictedwith a colon cancer-related disease (e.g., new detection (“screening”),detection of recurrence, reflex testing), the method comprisescomparing: a) the level of expression of a marker in a patient sample,and b) the normal level of expression of the marker in a controlnon-colon cancer-related disease sample. A significantly higher level ofexpression of the marker in the patient sample as compared to the normallevel is an indication that the patient is afflicted with a coloncancer-related disease.

There is also provided diagnostic methods for assessing the efficacy ofa therapy for inhibiting a colon cancer-related disease in a patient.Such methods comprise comparing: a) expression of a marker in a firstsample obtained from the patient prior to providing at least a portionof the therapy to the patient, and b) expression of the marker in asecond sample obtained from the patient following provision of theportion of the therapy. A significantly lower level of expression of themarker in the second sample relative to that in the first sample is anindication that the therapy is efficacious for inhibiting a coloncancer-related disease in the patient.

It will be appreciated that in these methods the “therapy” may be anytherapy for treating a colon cancer-related disease including, but notlimited to, pharmaceutical compositions, gene therapy and biologictherapy such as the administering of antibodies and chemokines. Thus,the methods described herein may be used to evaluate a patient before,during and after therapy, for example, to evaluate the reduction indisease state.

In certain aspects, the diagnostic methods are directed to therapy usinga chemical or biologic agent. These methods comprise comparing: a)expression of a marker in a first sample obtained from the patient andmaintained in the presence of the chemical or biologic agent, and b)expression of the marker in a second sample obtained from the patientand maintained in the absence of the agent. A significantly lower levelof expression of the marker in the second sample relative to that in thefirst sample is an indication that the agent is efficacious forinhibiting a colon cancer-related disease in the patient. In oneembodiment, the first and second samples can be portions of a singlesample obtained from the patient or portions of pooled samples obtainedfrom the patient.

There is also provided a monitoring method for assessing the progressionof a colon cancer-related disease in a patient, the method comprising:a) detecting in a patient sample at a first time point, the expressionof a marker; b) repeating step a) at a subsequent time point in time;and c) comparing the level of expression detected in steps a) and b),and therefrom monitoring the progression of a colon cancer-relateddisease in the patient. A significantly higher level of expression ofthe marker in the sample at the subsequent time point from that of thesample at the first time point is an indication that the coloncancer-related disease has progressed, whereas a significantly lowerlevel of expression is an indication that the colon cancer-relateddisease has regressed.

There is further provided a diagnostic method for determining whether acolon cancer-related disease has worsened or is likely to worsen in thefuture, the method comprising comparing: a) the level of expression of amarker in a patient sample, and b) the normal level of expression of themarker in a control sample. A significantly higher level of expressionin the patient sample as compared to the normal level is an indicationthat the colon cancer-related disease has worsened or is likely toworsen in the future.

There is also provided a test method for selecting a composition forinhibiting a colon cancer-related disease in a patient. This methodcomprises the steps of: a) obtaining a sample comprising cells from thepatient; b) separately maintaining aliquots of the sample in thepresence of a plurality of test compositions; c) comparing expression ofa marker in each of the aliquots; and d) selecting one of the testcompositions which significantly reduces the level of expression of themarker in the aliquot containing that test composition, relative to thelevels of expression of the marker in the presence of the other testcompositions.

There is additionally provided a test method of assessing the harmfulpotential of a compound in causing a colon cancer-related disease. Thismethod comprises the steps of: a) maintaining separate aliquots of cellsin the presence and absence of the compound; and b) comparing expressionof a marker in each of the aliquots. A significantly higher level ofexpression of the marker in the aliquot maintained in the presence ofthe compound, relative to that of the aliquot maintained in the absenceof the compound, is an indication that the compound possesses suchharmful potential.

In addition, there is further provided a method of inhibiting a coloncancer-related disease in a patient. This method comprises the steps of:a) obtaining a sample comprising cells from the patient; b) separatelymaintaining aliquots of the sample in the presence of a plurality ofcompositions; c) comparing expression of a marker in each of thealiquots; and d) administering to the patient at least one of thecompositions which significantly lowers the level of expression of themarker in the aliquot containing that composition, relative to thelevels of expression of the marker in the presence of the othercompositions.

The level of expression of a marker in a sample can be assessed, forexample, by detecting the presence in the sample of: the correspondingmarker protein or a fragment of the protein (e.g. by using a reagent,such as an antibody, an antibody derivative, an antibody fragment orsingle-chain antibody, which binds specifically with the protein orprotein fragment) the corresponding marker nucleic acid (e.g. anucleotide transcript, or a complement thereof), or a fragment of thenucleic acid (e.g. by contacting transcribed polynucleotides obtainedfrom the sample with a substrate having affixed thereto one or morenucleic acids having the entire or a segment of the nucleic acidsequence or a complement thereof) a metabolite which is produceddirectly (i.e., catalyzed) or indirectly by the corresponding markerprotein.

Any of the aforementioned methods may be performed using at least one ora plurality (e.g., 2, 3, 5, or 10 or more) of colon cancer-relateddisease markers, including colon cancer-related disease markers.

In such methods, the level of expression in the sample of each of aplurality of markers, at least one of which is a marker, is comparedwith the normal level of expression of each of the plurality of markersin samples of the same type obtained from control humans not afflictedwith a colon cancer-related disease. A significantly altered (i.e.,increased or decreased as specified in the above-described methods usinga single marker) level of expression in the sample of one or moremarkers, or some combination thereof, relative to that marker'scorresponding normal or control level, is an indication that the patientis afflicted with a colon cancer-related disease. For all of theaforementioned methods, the marker(s) are selected such that thepositive predictive value of the method is at least about 10%.

In another aspect, there is provided various diagnostic and test kits.In one embodiment, a kit is useful for assessing whether a patient isafflicted with a colon cancer-related disease. The kit comprises areagent for assessing expression of a marker. In another embodiment, akit is useful for assessing the suitability of a chemical or biologicagent for inhibiting a colon cancer-related disease in a patient. Such akit comprises a reagent for assessing expression of a marker, and mayalso comprise one or more of such agents.

In a further embodiment, the kits are useful for assessing the presenceof colon cancer-related disease cells or treating colon cancer-relateddiseases. Such kits comprise an antibody, an antibody derivative or anantibody fragment, which binds specifically with a marker protein or afragment of the protein. Such kits may also comprise a plurality ofantibodies, antibody derivatives or antibody fragments wherein theplurality of such antibody agents binds specifically with a markerprotein or a fragment of the protein.

In an additional embodiment, the kits are useful for assessing thepresence of colon cancer-related disease cells, wherein the kitcomprises a nucleic acid probe that binds specifically with a markernucleic acid or a fragment of the nucleic acid. The kit may alsocomprise a plurality of probes, wherein each of the probes bindsspecifically with a marker nucleic acid, or a fragment of the nucleicacid.

In a further aspect, there is provided methods for treating a patientafflicted with a colon cancer-related disease or at risk of developing acolon cancer-related disease. Such methods may comprise reducing theexpression and/or interfering with the biological function of a marker.In one embodiment, the method comprises providing to the patient anantisense oligonucleotide or polynucleotide complementary to a markernucleic acid, or a segment thereof. For example, an antisensepolynucleotide may be provided to the patient through the delivery of avector that expresses an anti-sense polynucleotide of a marker nucleicacid or a fragment thereof. In another embodiment, the method comprisesproviding to the patient an antibody, an antibody derivative or antibodyfragment, which binds specifically with a marker protein, or a fragmentof the protein.

In a broad aspect, there is provided a method for producing a non-humananimal model for assessment of at least one colon cancer-relateddisease. The method includes exposing the animal to repeated doses of atleast one chemical believed to cause colon cancer. In certain aspects,the method further includes collecting one or more selected samples fromthe animal; and comparing the collected sample to one or more indicia ofpotential colon cancer initiation or development.

In broad aspect, there is provides a method of producing the animalmodel that includes: maintaining the animal in a specific chemical-freeenvironment and sensitizing the animal with at least one chemicalbelieved to cause colon cancer. In certain embodiments, at least a partof the animal's colon is sensitized by multiple sequential exposures.

In another broad aspect, there is provided a method of screening for anagent for effectiveness against at least one colon cancer-relateddisease. The method generally includes: administering at least one agentto the animal, determining whether the agent reduces or aggravates oneor more symptoms of the colon cancer-related disease; correlating areduction in one or more symptoms with effectiveness of the agentagainst the colon cancer-related disease; or correlating a lack ofreduction in one or more symptoms with ineffectiveness of the agent.

The animal model is useful for assessing one or more metabolic pathwaysthat contribute to at least one of initiation, progression, severity,pathology, aggressiveness, grade, activity, disability, mortality,morbidity, disease sub-classification or other underlying pathogenic orpathological feature of at least one colon cancer-related disease. Theanalysis can be by one or more of: hierarchical clustering, signaturenetwork construction, mass spectroscopy proteomic analysis, surfaceplasmon resonance, linear statistical modeling, partial least squaresdiscriminant analysis, and multiple linear regression analysis.

In a particular aspect, the animal model is assessed for at least onecolon cancer-related disease, by examining an expression level of one ormore markers, or a functional equivalent thereto.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods whichare within the skill of the art. Such techniques are explained fully inthe literature. See, for example, Molecular Cloning A Laboratory Manual,2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (Glover ed.,1985); Oligonucleotide Synthesis (Gait ed., 1984); Mullis et al. U.S.Pat. No. 4,683,195; Nucleic Acid Hybridization (Hames & Higgins eds.,1984); Transcription And Translation (Hames & Higgins eds., 1984);Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A PracticalGuide To Molecular Cloning (1984); the treatise, Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells(Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weirand Blackwell, eds., 1986); The Laboratory Rat, editor in chief: Mark A.Suckow; authors: Sharp and LaRegina. CRC Press, Boston, 1988, which areincorporated herein by reference) and chemical methods.

Described herein are newly discovered markers associated with a coloncancer-induced state of various cells. It has been discovered that thehigher than normal level of expression of any of these markers orcombination of these markers correlates with the presence of a coloncancer-related disease in a patient. Methods are provided for detectingthe presence of a colon cancer-related disease in a sample; the absenceof a in a sample; the stage of a colon cancer-related disease; and,other characteristics of a colon cancer-related disease that arerelevant to the assessment, prevention, diagnosis, characterization andtherapy of a colon cancer-related disease in a patient. Methods oftreating a colon cancer-related disease are also provided.

Definitions As used herein, each of the following terms has the meaningassociated with it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

A “marker” is a gene or protein whose altered level of expression in atissue or cell from its expression level in normal or healthy tissue orcell is associated with a disease state.

The “normal” level of expression of a marker is the level of expressionof the marker in colon system cells of a human subject or patient notafflicted with a colon cancer-related disease.

An “over-expression” or “significantly higher level of expression” of amarker refers to an expression level in a test sample that is greaterthan the standard error of the assay employed to assess expression, andin certain embodiments, at least twice, and in other embodiments, three,four, five or ten times the expression level of the marker in a controlsample (e.g., sample from a healthy subject not having the markerassociated disease) and in certain embodiments, the average expressionlevel of the marker in several control samples.

A “significantly lower level of expression” of a marker refers to anexpression level in a test sample that is at least twice, and in certainembodiments, three, four, five or ten times lower than the expressionlevel of the marker in a control sample (e.g., sample from a healthysubject not having the marker associated disease) and in certainembodiments, the average expression level of the marker in severalcontrol samples.

A kit is any manufacture (e.g. a package or container) comprising atleast one reagent, e.g., a probe, for specifically detecting theexpression of a marker. The kit may be promoted, distributed or sold asa unit for performing the methods of the present invention.

“Proteins” encompass marker proteins and their fragments; variant markerproteins and their fragments; peptides and polypeptides comprising an atleast 15 amino acid segment of a marker or variant marker protein; andfusion proteins comprising a marker or variant marker protein, or an atleast 15 amino acid segment of a marker or variant marker protein.

The compositions, kits and methods described herein have the followinguses, among others: 1) assessing whether a patient is afflicted with acolon cancer-related disease; 2) assessing the stage of a coloncancer-related disease in a human patient; 3) assessing the grade of acolon cancer-related disease in a patient; 4) assessing the nature of acolon cancer-related disease in a patient; 5) assessing the potential todevelop a colon cancer-related disease in a patient; 6) assessing thehistological type of cells associated with a colon cancer-relateddisease in a patient; 7) making antibodies, antibody fragments orantibody derivatives that are useful for treating a colon cancer-relateddisease and/or assessing whether a patient is afflicted with a coloncancer-related disease; 8) assessing the presence of coloncancer-related disease cells; 9) assessing the efficacy of one or moretest compounds for inhibiting a colon cancer-related disease in apatient; 10) assessing the efficacy of a therapy for inhibiting a coloncancer-related disease in a patient; 11) monitoring the progression of acolon cancer-related disease in a patient; 12) selecting a compositionor therapy for inhibiting a colon cancer-related disease in a patient;13) treating a patient afflicted with a colon cancer-related disease;14) inhibiting a colon cancer-related disease in a patient; 15)assessing the harmful potential of a test compound; and 16) preventingthe onset of a colon cancer-related disease in a patient at risk fordeveloping a colon cancer-related disease.

Screening Methods

The animal models created by the methods described herein will enablescreening of therapeutic agents useful for treating or preventing acolon cancer-related disease. Accordingly, the methods are useful foridentifying therapeutic agents for treating or preventing a coloncancer-related disease. The methods comprise administering a candidateagent to an animal model made by the methods described herein, assessingat least one colon cancer-related disease response in the animal modelas compared to a control animal model to which the candidate agent hasnot been administered. If at least one colon cancer-related diseaseresponse is reduced in symptoms or delayed in onset, the candidate agentis an agent for treating or preventing the colon cancer-related disease.

The candidate agents may be pharmacologic agents already known in theart or may be agents previously unknown to have any pharmacologicalactivity. The agents may be naturally arising or designed in thelaboratory. They may be isolated from microorganisms, animals or plants,or may be produced recombinantly, or synthesized by any suitablechemical method. They may be small molecules, nucleic acids, proteins,peptides or peptidomimetics. In certain embodiments, candidate agentsare small organic compounds having a molecular weight of more than 50and less than about 2,500 daltons. Candidate agents comprise functionalgroups necessary for structural interaction with proteins. Candidateagents are also found among biomolecules including, but not limited to:peptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. There are, for example,numerous means available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. In certain embodiments, thecandidate agents can be obtained using any of the numerous approaches incombinatorial library methods art, including, by non-limiting example:biological libraries; spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection.

In certain further embodiments, certain pharmacological agents may besubjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

The same methods for identifying therapeutic agents for treating a coloncancer-related disease can also be used to validate leadcompounds/agents generated from in vitro studies.

The candidate agent may be an agent that up- or down-regulates one ormore colon cancer-related disease response pathways. In certainembodiments, the candidate agent may be an antagonist that affects suchpathway.

Methods for Treating a Colon Cancer-Related Disease

There is provided herein methods for treating, inhibiting, relieving orreversing a colon cancer-related disease response. In the methodsdescribed herein, an agent that interferes with a signaling cascade isadministered to an individual in need thereof, such as, but not limitedto, colon cancer-related disease patients in whom such complications arenot yet evident and those who already have at least one coloncancer-related disease response.

In the former instance, such treatment is useful to prevent theoccurrence of such colon cancer-related disease response and/or reducethe extent to which they occur. In the latter instance, such treatmentis useful to reduce the extent to which such colon cancer-relateddisease response occurs, prevent their further development or reversethe colon cancer-related disease response.

In certain embodiments, the agent that interferes with the coloncancer-related disease response cascade may be an antibody specific forsuch response.

Expression of a Marker

Expression of a marker can be inhibited in a number of ways, including,by way of a non-limiting example, an antisense oligonucleotide can beprovided to the colon cancer-related disease cells in order to inhibittranscription, translation, or both, of the marker(s). Alternately, apolynucleotide encoding an antibody, an antibody derivative, or anantibody fragment which specifically binds a marker protein, andoperably linked with an appropriate promoter/regulator region, can beprovided to the cell in order to generate intracellular antibodies whichwill inhibit the function or activity of the protein. The expressionand/or function of a marker may also be inhibited by treating the coloncancer-related disease cell with an antibody, antibody derivative orantibody fragment that specifically binds a marker protein. Using themethods described herein, a variety of molecules, particularly includingmolecules sufficiently small that they are able to cross the cellmembrane, can be screened in order to identify molecules which inhibitexpression of a marker or inhibit the function of a marker protein. Thecompound so identified can be provided to the patient in order toinhibit colon cancer-related disease cells of the patient.

Any marker or combination of markers, as well as any certain markers incombination with the markers, may be used in the compositions, kits andmethods described herein. In general, it is desirable to use markers forwhich the difference between the level of expression of the marker incolon cancer-related disease cells and the level of expression of thesame marker in normal colon system cells is as great as possible.Although this difference can be as small as the limit of detection ofthe method for assessing expression of the marker, it is desirable thatthe difference be at least greater than the standard error of theassessment method, and, in certain embodiments, a difference of at least2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 100-, 500-, 1000-fold orgreater than the level of expression of the same marker in normaltissue.

It is recognized that certain marker proteins are secreted to theextracellular space surrounding the cells. These markers are used incertain embodiments of the compositions, kits and methods, owing to thefact that such marker proteins can be detected in a coloncancer-associated body fluid sample, which may be more easily collectedfrom a human patient than a tissue biopsy sample. In addition, in vivotechniques for detection of a marker protein include introducing into asubject a labeled antibody directed against the protein. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In order to determine whether any particular marker protein is asecreted protein, the marker protein is expressed in, for example, amammalian cell, such as a human colon line, extracellular fluid iscollected, and the presence or absence of the protein in theextracellular fluid is assessed (e.g. using a labeled antibody whichbinds specifically with the protein).

It will be appreciated that patient samples containing colon cells maybe used in the methods described herein. In these embodiments, the levelof expression of the marker can be assessed by assessing the amount(e.g., absolute amount or concentration) of the marker in a sample. Thecell sample can, of course, be subjected to a variety of post-collectionpreparative and storage techniques (e.g., nucleic acid and/or proteinextraction, fixation, storage, freezing, ultrafiltration, concentration,evaporation, centrifugation, etc.) prior to assessing the amount of themarker in the sample.

It will also be appreciated that the markers may be shed from the cellsinto the digestive system, the blood stream and/or interstitial spaces.The shed markers can be tested, for example, by examining the serum orplasma.

The compositions, kits and methods can be used to detect expression ofmarker proteins having at least one portion which is displayed on thesurface of cells which express it. For example, immunological methodsmay be used to detect such proteins on whole cells, or computer-basedsequence analysis methods may be used to predict the presence of atleast one extracellular domain (i.e., including both secreted proteinsand proteins having at least one cell-surface domain). Expression of amarker protein having at least one portion which is displayed on thesurface of a cell which expresses it may be detected without necessarilylysing the cell (e.g., using a labeled antibody which binds specificallywith a cell-surface domain of the protein).

Expression of a marker may be assessed by any of a wide variety ofmethods for detecting expression of a transcribed nucleic acid orprotein. Non-limiting examples of such methods include immunologicalmethods for detection of secreted, cell-surface, cytoplasmic or nuclearproteins, protein purification methods, protein function or activityassays, nucleic acid hybridization methods, nucleic acid reversetranscription methods and nucleic acid amplification methods.

In a particular embodiment, expression of a marker is assessed using anantibody (e.g., a radio-labeled, chromophore-labeled,fluorophore-labeled or enzyme-labeled antibody), an antibody derivative(e.g., an antibody conjugated with a substrate or with the protein orligand of a protein-ligand pair), or an antibody fragment (e.g., asingle-chain antibody, an isolated antibody hypervariable domain, etc.)which binds specifically with a marker protein or fragment thereof,including a marker protein which has undergone all or a portion of itsnormal post-translational modification.

In another particular embodiment, expression of a marker is assessed bypreparing mRNA/cDNA (i.e., a transcribed polynucleotide) from cells in apatient sample, and by hybridizing the mRNA/cDNA with a referencepolynucleotide which is a complement of a marker nucleic acid, or afragment thereof. cDNA can, optionally, be amplified using any of avariety of polymerase chain reaction methods prior to hybridization withthe reference polynucleotide; preferably, it is not amplified.Expression of one or more markers can likewise be detected usingquantitative PCR to assess the level of expression of the marker(s).Alternatively, any of the many methods of detecting mutations orvariants (e.g., single nucleotide polymorphisms, deletions, etc.) of amarker may be used to detect occurrence of a marker in a patient.

In a related embodiment, a mixture of transcribed polynucleotidesobtained from the sample is contacted with a substrate having fixedthereto a polynucleotide complementary to or homologous with at least aportion (e.g., at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or morenucleotide residues) of a marker nucleic acid. If polynucleotidescomplementary to or homologous with are differentially detectable on thesubstrate (e.g., detectable using different chromophores orfluorophores, or fixed to different selected positions), then the levelsof expression of a plurality of markers can be assessed simultaneouslyusing a single substrate (e.g., a “gene chip” microarray ofpolynucleotides fixed at selected positions). When a method of assessingmarker expression is used which involves hybridization of one nucleicacid with another, it is desired that the hybridization be performedunder stringent hybridization conditions.

In certain embodiments, the biomarker assays can be performed using massspectrometry or surface plasmon resonance. In various embodiment, themethod of identifying an agent active against a colon cancer-relateddisease can include a) providing a sample of cells containing one ormore markers or derivative thereof; b) preparing an extract from saidcells; c) mixing said extract with a labeled nucleic acid probecontaining a marker binding site; and, d) determining the formation of acomplex between the marker and the nucleic acid probe in the presence orabsence of the test agent. The determining step can include subjectingsaid extract/nucleic acid probe mixture to an electrophoretic mobilityshift assay.

In certain embodiments, the determining step comprises an assay selectedfrom an enzyme linked immunoabsorption assay (ELISA), fluorescence basedassays and ultra high throughput assays, for example surface plasmonresonance (SPR) or fluorescence correlation spectroscopy (FCS) assays.In such embodiments, the SPR sensor is useful for direct real-timeobservation of biomolecular interactions since SPR is sensitive tominute refractive index changes at a metal-dielectric surface. SPR is asurface technique that is sensitive to changes of 10⁵ to 10⁻⁶ refractiveindex (RI) units within approximately 200 nm of the SPR sensor/sampleinterface. Thus, SPR spectroscopy is useful for monitoring the growth ofthin organic films deposited on the sensing layer.

Because the compositions, kits, and methods rely on detection of adifference in expression levels of one or more markers, it is desiredthat the level of expression of the marker is significantly greater thanthe minimum detection limit of the method used to assess expression inat least one of normal cells and colon cancer-affected cells.

It is understood that by routine screening of additional patient samplesusing one or more of the markers, it will be realized that certain ofthe markers are over-expressed in cells of various types, includingspecific colon cancer-related diseases.

In addition, as a greater number of patient samples are assessed forexpression of the markers and the outcomes of the individual patientsfrom whom the samples were obtained are correlated, it will also beconfirmed that altered expression of certain of the markers are stronglycorrelated with a colon cancer-related disease and that alteredexpression of other markers are strongly correlated with other diseases.The compositions, kits, and methods are thus useful for characterizingone or more of the stage, grade, histological type, and nature of acolon cancer-related disease in patients.

When the compositions, kits, and methods are used for characterizing oneor more of the stage, grade, histological type, and nature of a coloncancer-related disease in a patient, it is desired that the marker orpanel of markers is selected such that a positive result is obtained inat least about 20%, and in certain embodiments, at least about 40%, 60%,or 80%, and in substantially all patients afflicted with a coloncancer-related disease of the corresponding stage, grade, histologicaltype, or nature. The marker or panel of markers invention can beselected such that a positive predictive value of greater than about 10%is obtained for the general population (in a non-limiting example,coupled with an assay specificity greater than 80%).

When a plurality of markers are used in the compositions, kits, andmethods, the level of expression of each marker in a patient sample canbe compared with the normal level of expression of each of the pluralityof markers in non-colon cancer samples of the same type, either in asingle reaction mixture (i.e. using reagents, such as differentfluorescent probes, for each marker) or in individual reaction mixturescorresponding to one or more of the markers. In one embodiment, asignificantly increased level of expression of more than one of theplurality of markers in the sample, relative to the corresponding normallevels, is an indication that the patient is afflicted with a coloncancer-related disease. When a plurality of markers is used, 2, 3, 4, 5,8, 10, 12, 15, 20, 30, or 50 or more individual markers can be used; incertain embodiments, the use of fewer markers may be desired.

In order to maximize the sensitivity of the compositions, kits, andmethods (i.e. by interference attributable to cells of non-colon systemorigin in a patient sample), it is desirable that the marker usedtherein be a marker which has a restricted tissue distribution, e.g.,normally not expressed in a non-colon system tissue.

It is recognized that the compositions, kits, and methods will be ofparticular utility to patients having an enhanced risk of developing acolon cancer-related disease and their medical advisors. Patientsrecognized as having an enhanced risk of developing a coloncancer-related disease include, for example, patients having a familialhistory of a colon cancer-related disease.

The level of expression of a marker in normal human colon system tissuecan be assessed in a variety of ways. In one embodiment, this normallevel of expression is assessed by assessing the level of expression ofthe marker in a portion of colon system cells which appear to be normaland by comparing this normal level of expression with the level ofexpression in a portion of the colon system cells which is suspected ofbeing abnormal. Alternately, and particularly as further informationbecomes available as a result of routine performance of the methodsdescribed herein, population-average values for normal expression of themarkers may be used. In other embodiments, the ‘normal’ level ofexpression of a marker may be determined by assessing expression of themarker in a patient sample obtained from a non-colon cancer-afflictedpatient, from a patient sample obtained from a patient before thesuspected onset of a colon cancer-related disease in the patient, fromarchived patient samples, and the like.

There is also provided herein compositions, kits, and methods forassessing the presence of colon cancer-related disease cells in a sample(e.g. an archived tissue sample or a sample obtained from a patient).These compositions, kits, and methods are substantially the same asthose described above, except that, where necessary, the compositions,kits, and methods are adapted for use with samples other than patientsamples. For example, when the sample to be used is a parafinized,archived human tissue sample, it can be necessary to adjust the ratio ofcompounds in the compositions, in the kits, or the methods used toassess levels of marker expression in the sample.

Kits and Reagents

The kits are useful for assessing the presence of colon cancer-relateddisease cells (e.g. in a sample such as a patient sample). The kitcomprises a plurality of reagents, each of which is capable of bindingspecifically with a marker nucleic acid or protein. Suitable reagentsfor binding with a marker protein include antibodies, antibodyderivatives, antibody fragments, and the like. Suitable reagents forbinding with a marker nucleic acid (e.g. a genomic DNA, an mRNA, aspliced mRNA, a cDNA, or the like) include complementary nucleic acids.For example, the nucleic acid reagents may include oligonucleotides(labeled or non-labeled) fixed to a substrate, labeled oligonucleotidesnot bound with a substrate, pairs of PCR primers, molecular beaconprobes, and the like.

The kits may optionally comprise additional components useful forperforming the methods described herein. By way of example, the kit maycomprise fluids (e.g. SSC buffer) suitable for annealing complementarynucleic acids or for binding an antibody with a protein with which itspecifically binds, one or more sample compartments, an instructionalmaterial which describes performance of the method, a sample of normalcolon system cells, a sample of colon cancer-related disease cells, andthe like.

Method of Producing Antibodies

There is also provided herein a method of making an isolated hybridomawhich produces an antibody useful for assessing whether a patient isafflicted with a colon cancer-related disease. In this method, a proteinor peptide comprising the entirety or a segment of a marker protein issynthesized or isolated (e.g. by purification from a cell in which it isexpressed or by transcription and translation of a nucleic acid encodingthe protein or peptide in vivo or in vitro). A vertebrate, for example,a mammal such as a mouse, rat, rabbit, or sheep, is immunized using theprotein or peptide. The vertebrate may optionally (and preferably) beimmunized at least one additional time with the protein or peptide, sothat the vertebrate exhibits a robust immune response to the protein orpeptide. Splenocytes are isolated from the immunized vertebrate andfused with an immortalized cell line to form hybridomas, using any of avariety of methods. Hybridomas formed in this manner are then screenedusing standard methods to identify one or more hybridomas which producean antibody which specifically binds with the marker protein or afragment thereof. There is also provided herein hybridomas made by thismethod and antibodies made using such hybridomas.

Method of Assessing Efficacy

There is also provided herein a method of assessing the efficacy of atest compound for inhibiting colon cancer-related disease cells. Asdescribed above, differences in the level of expression of the markerscorrelate with the abnormal state of colon system cells. Although it isrecognized that changes in the levels of expression of certain of themarkers likely result from the abnormal state of colon system cells, itis likewise recognized that changes in the levels of expression of otherof the markers induce, maintain, and promote the abnormal state of thosecells. Thus, compounds which inhibit a colon cancer-related disease in apatient will cause the level of expression of one or more of the markersto change to a level nearer the normal level of expression for thatmarker (i.e. the level of expression for the marker in normal colonsystem cells).

This method thus comprises comparing expression of a marker in a firstcolon cell sample and maintained in the presence of the test compoundand expression of the marker in a second colon cell sample andmaintained in the absence of the test compound. A significantly reducedexpression of a marker in the presence of the test compound is anindication that the test compound inhibits a colon cancer-relateddisease. The colon cell samples may, for example, be aliquots of asingle sample of normal colon cells obtained from a patient, pooledsamples of normal colon cells obtained from a patient, cells of a normalcolon cell line, aliquots of a single sample of colon cancer-relateddisease cells obtained from a patient, pooled samples of coloncancer-related disease cells obtained from a patient, cells of a coloncancer-related disease cell line, or the like.

In one embodiment, the samples are colon cancer-related disease cellsobtained from a patient and a plurality of compounds believed to beeffective for inhibiting various colon cancer-related diseases aretested in order to identify the compound which is likely to best inhibitthe colon cancer-related disease in the patient.

This method may likewise be used to assess the efficacy of a therapy forinhibiting a colon cancer-related disease in a patient. In this method,the level of expression of one or more markers in a pair of samples (onesubjected to the therapy, the other not subjected to the therapy) isassessed. As with the method of assessing the efficacy of testcompounds, if the therapy induces a significantly lower level ofexpression of a marker then the therapy is efficacious for inhibiting acolon cancer-related disease. As above, if samples from a selectedpatient are used in this method, then alternative therapies can beassessed in vitro in order to select a therapy most likely to beefficacious for inhibiting a colon cancer-related disease in thepatient.

As described herein, the abnormal state of human colon cells iscorrelated with changes in the levels of expression of the markers.There is also provided a method for assessing the harmful potential of atest compound. This method comprises maintaining separate aliquots ofhuman colon cells in the presence and absence of the test compound.Expression of a marker in each of the aliquots is compared. Asignificantly higher level of expression of a marker in the aliquotmaintained in the presence of the test compound (relative to the aliquotmaintained in the absence of the test compound) is an indication thatthe test compound possesses a harmful potential. The relative harmfulpotential of various test compounds can be assessed by comparing thedegree of enhancement or inhibition of the level of expression of therelevant markers, by comparing the number of markers for which the levelof expression is enhanced or inhibited, or by comparing both.

Various aspects are described in further detail in the followingsubsections.

Isolated Proteins and Antibodies

One aspect pertains to isolated marker proteins and biologically activeportions thereof, as well as polypeptide fragments suitable for use asimmunogens to raise antibodies directed against a marker protein or afragment thereof. In one embodiment, the native marker protein can beisolated from cells or tissue sources by an appropriate purificationscheme using standard protein purification techniques. In anotherembodiment, a protein or peptide comprising the whole or a segment ofthe marker protein is produced by recombinant DNA techniques.Alternative to recombinant expression, such protein or peptide can besynthesized chemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”).

When the protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, 10%, or 5% of thevolume of the protein preparation. When the protein is produced bychemical synthesis, it is preferably substantially free of chemicalprecursors or other chemicals, i.e., it is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. Accordingly such preparations of the protein have less thanabout 30%, 20%, 10%, 5% (by dry weight) of chemical precursors orcompounds other than the polypeptide of interest.

Biologically active portions of a marker protein include polypeptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the marker protein, which include feweramino acids than the full length protein, and exhibit at least oneactivity of the corresponding full-length protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the corresponding full-length protein. A biologicallyactive portion of a marker protein can be a polypeptide which is, forexample, 10, 25, 50, 100 or more amino acids in length. Moreover, otherbiologically active portions, in which other regions of the markerprotein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of the nativeform of the marker protein. In certain embodiments, useful proteins aresubstantially identical (e.g., at least about 40%, and in certainembodiments, 50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of thesesequences and retain the functional activity of the correspondingnaturally-occurring marker protein yet differ in amino acid sequence dueto natural allelic variation or mutagenesis.

In addition, libraries of segments of a marker protein can be used togenerate a variegated population of polypeptides for screening andsubsequent selection of variant marker proteins or segments thereof.

Predictive Medicine

There is also provided herein uses of the animal models and markers inthe field of predictive medicine in which diagnostic assays, prognosticassays, pharmacogenomics, and monitoring clinical trials are used forprognostic (predictive) purposes to thereby treat an individualprophylactically. Accordingly, there is also provided herein diagnosticassays for determining the level of expression of one or more markerproteins or nucleic acids, in order to determine whether an individualis at risk of developing a colon cancer-related disease. Such assays canbe used for prognostic or predictive purposes to therebyprophylactically treat an individual prior to the onset of the coloncancer-related disease.

In another aspect, the methods are useful for at least periodicscreening of the same individual to see if that individual has beenexposed to chemicals or toxins that change his/her expression patterns.

Yet another aspect pertains to monitoring the influence of agents (e.g.,drugs or other compounds administered either to inhibit a coloncancer-related disease or to treat or prevent any other disorder (e.g.,in order to understand any system effects that such treatment may have)on the expression or activity of a marker in clinical trials.

Pharmacogenomics

The markers are also useful as pharmacogenomic markers. As used herein,a “pharmacogenomic marker” is an objective biochemical marker whoseexpression level correlates with a specific clinical drug response orsusceptibility in a patient. The presence or quantity of thepharmacogenomic marker expression is related to the predicted responseof the patient and more particularly the patient's tumor to therapy witha specific drug or class of drugs. By assessing the presence or quantityof the expression of one or more pharmacogenomic markers in a patient, adrug therapy which is most appropriate for the patient, or which ispredicted to have a greater degree of success, may be selected.

Monitoring Clinical Trials

Monitoring the influence of agents (e.g., drug compounds) on the levelof expression of a marker can be applied not only in basic drugscreening, but also in clinical trials. For example, the effectivenessof an agent to affect marker expression can be monitored in clinicaltrials of subjects receiving treatment for a colon cancer-relateddisease.

In one non-limiting embodiment, the present invention provides a methodfor monitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate) comprising the steps of(i) obtaining a pre-administration sample from a subject prior toadministration of the agent; (ii) detecting the level of expression ofone or more selected markers in the pre-administration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression of the marker(s) in thepost-administration samples; (v) comparing the level of expression ofthe marker(s) in the pre-administration sample with the level ofexpression of the marker(s) in the post-administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly.

For example, increased expression of the marker gene(s) during thecourse of treatment may indicate ineffective dosage and the desirabilityof increasing the dosage. Conversely, decreased expression of the markergene(s) may indicate efficacious treatment and no need to change dosage.

Electronic Apparatus Readable Media, Systems, Arrays and Methods ofUsing Same

As used herein, “electronic apparatus readable media” refers to anysuitable medium for storing, holding or containing data or informationthat can be read and accessed directly by an electronic apparatus. Suchmedia can include, but are not limited to: magnetic storage media, suchas floppy discs, hard disc storage medium, and magnetic tape; opticalstorage media such as compact disc; electronic storage media such asRAM, ROM, EPROM, EEPROM and the like; and general hard disks and hybridsof these categories such as magnetic/optical storage media. The mediumis adapted or configured for having recorded thereon a marker asdescribed herein.

As used herein, the term “electronic apparatus” is intended to includeany suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as personal digital assistants(PDAs), cellular phone, pager and the like; and local and distributedprocessing systems.

As used herein, “recorded” refers to a process for storing or encodinginformation on the electronic apparatus readable medium. Those skilledin the art can readily adopt any method for recording information onmedia to generate materials comprising the markers described herein.

A variety of software programs and formats can be used to store themarker information of the present invention on the electronic apparatusreadable medium. Any number of data processor structuring formats (e.g.,text file or database) may be employed in order to obtain or create amedium having recorded thereon the markers. By providing the markers inreadable form, one can routinely access the marker sequence informationfor a variety of purposes. For example, one skilled in the art can usethe nucleotide or amino acid sequences in readable form to compare atarget sequence or target structural motif with the sequence informationstored within the data storage means. Search means are used to identifyfragments or regions of the sequences which match a particular targetsequence or target motif.

Thus, there is also provided herein a medium for holding instructionsfor performing a method for determining whether a subject has a coloncancer-related disease or a pre-disposition to a colon cancer-relateddisease, wherein the method comprises the steps of determining thepresence or absence of a marker and based on the presence or absence ofthe marker, determining whether the subject has a colon cancer-relateddisease or a pre-disposition to a colon cancer-related disease and/orrecommending a particular treatment for a colon cancer-related diseaseor pre-colon cancer-related disease condition.

There is also provided herein an electronic system and/or in a network,a method for determining whether a subject has a colon cancer-relateddisease or a pre-disposition to a colon cancer-related diseaseassociated with a marker wherein the method comprises the steps ofdetermining the presence or absence of the marker, and based on thepresence or absence of the marker, determining whether the subject has acolon cancer-related disease or a pre-disposition to a coloncancer-related disease, and/or recommending a particular treatment forthe colon cancer-related disease or pre-colon cancer-related diseasecondition. The method may further comprise the step of receivingphenotypic information associated with the subject and/or acquiring froma network phenotypic information associated with the subject.

Also provided herein is a network, a method for determining whether asubject has a colon cancer-related disease or a pre-disposition to acolon cancer-related disease associated with a marker, the methodcomprising the steps of receiving information associated with themarker, receiving phenotypic information associated with the subject,acquiring information from the network corresponding to the markerand/or a colon cancer-related disease, and based on one or more of thephenotypic information, the marker, and the acquired information,determining whether the subject has a colon cancer-related disease or apre-disposition to a colon cancer-related disease. The method mayfurther comprise the step of recommending a particular treatment for thecolon cancer-related disease or pre-colon cancer-related diseasecondition.

There is also provided herein a business method for determining whethera subject has a colon cancer-related disease or a pre-disposition to acolon cancer-related disease, the method comprising the steps ofreceiving information associated with the marker, receiving phenotypicinformation associated with the subject, acquiring information from thenetwork corresponding to the marker and/or a colon cancer-relateddisease, and based on one or more of the phenotypic information, themarker, and the acquired information, determining whether the subjecthas a colon cancer-related disease or a pre-disposition to a coloncancer-related disease. The method may further comprise the step ofrecommending a particular treatment for the colon cancer-related diseaseor pre-colon cancer-related disease condition.

There is also provided herein an array that can be used to assayexpression of one or more genes in the array. In one embodiment, thearray can be used to assay gene expression in a tissue to ascertaintissue specificity of genes in the array. In this manner, up to about7000 or more genes can be simultaneously assayed for expression. Thisallows a profile to be developed showing a battery of genes specificallyexpressed in one or more tissues.

In addition to such qualitative determination, there is provided hereinthe quantitation of gene expression. Thus, not only tissue specificity,but also the level of expression of a battery of genes in the tissue isascertainable. Thus, genes can be grouped on the basis of their tissueexpression per se and level of expression in that tissue. This isuseful, for example, in ascertaining the relationship of gene expressionbetween or among tissues. Thus, one tissue can be perturbed and theeffect on gene expression in a second tissue can be determined. In thiscontext, the effect of one cell type on another cell type in response toa biological stimulus can be determined.

Such a determination is useful, for example, to know the effect ofcell-cell interaction at the level of gene expression. If an agent isadministered therapeutically to treat one cell type but has anundesirable effect on another cell type, the method provides an assay todetermine the molecular basis of the undesirable effect and thusprovides the opportunity to co-administer a counteracting agent orotherwise treat the undesired effect. Similarly, even within a singlecell type, undesirable biological effects can be determined at themolecular level. Thus, the effects of an agent on expression of otherthan the target gene can be ascertained and counteracted.

In another embodiment, the array can be used to monitor the time courseof expression of one or more genes in the array. This can occur invarious biological contexts, as disclosed herein, for exampledevelopment of a colon cancer-related disease, progression of a coloncancer-related disease, and processes, such as cellular transformationassociated with a colon cancer-related disease.

The array is also useful for ascertaining the effect of the expressionof a gene or the expression of other genes in the same cell or indifferent cells. This provides, for example, for a selection ofalternate molecular targets for therapeutic intervention if the ultimateor downstream target cannot be regulated.

The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes that could serve as a molecular target fordiagnosis or therapeutic intervention.

Surrogate Markers

The markers may serve as surrogate markers for one or more disorders ordisease states or for conditions leading up to a colon cancer-relateddisease state. As used herein, a “surrogate marker” is an objectivebiochemical marker which correlates with the absence or presence of adisease or disorder, or with the progression of a disease or disorder.The presence or quantity of such markers is independent of the disease.Therefore, these markers may serve to indicate whether a particularcourse of treatment is effective in lessening a disease state ordisorder. Surrogate markers are of particular use when the presence orextent of a disease state or disorder is difficult to assess throughstandard methodologies, or when an assessment of disease progression isdesired before a potentially dangerous clinical endpoint is reached.

The markers are also useful as pharmacodynamic markers. As used herein,a “pharmacodynamic marker” is an objective biochemical marker whichcorrelates specifically with drug effects. The presence or quantity of apharmacodynamic marker is not related to the disease state or disorderfor which the drug is being administered; therefore, the presence orquantity of the marker is indicative of the presence or activity of thedrug in a subject. For example, a pharmacodynamic marker may beindicative of the concentration of the drug in a biological tissue, inthat the marker is either expressed or transcribed or not expressed ortranscribed in that tissue in relationship to the level of the drug. Inthis fashion, the distribution or uptake of the drug may be monitored bythe pharmacodynamic marker. Similarly, the presence or quantity of thepharmacodynamic marker may be related to the presence or quantity of themetabolic product of a drug, such that the presence or quantity of themarker is indicative of the relative breakdown rate of the drug in vivo.

Pharmacodynamic markers are of particular use in increasing thesensitivity of detection of drug effects, particularly when the drug isadministered in low doses. Since even a small amount of a drug may besufficient to activate multiple rounds of marker transcription orexpression, the amplified marker may be in a quantity which is morereadily detectable than the drug itself. Also, the marker may be moreeasily detected due to the nature of the marker itself; for example,using the methods described herein, antibodies may be employed in animmune-based detection system for a protein marker, or marker-specificradiolabeled probes may be used to detect a mRNA marker. Furthermore,the use of a pharmacodynamic marker may offer mechanism-based predictionof risk due to drug treatment beyond the range of possible directobservations.

Protocols for Testing

The method of testing for colon cancer-related diseases comprises, forexample measuring the expression level of each marker gene in abiological sample from a subject over time and comparing the level withthat of the marker gene in a control biological sample.

When the marker gene is one of the genes described herein and theexpression level is differentially expressed (for examples, higher orlower than that in the control), the subject is judged to be affectedwith a colon cancer-related disease. When the expression level of themarker gene falls within the permissible range, the subject is unlikelyto be affected with a colon cancer-related disease.

The standard value for the control may be pre-determined by measuringthe expression level of the marker gene in the control, in order tocompare the expression levels. For example, the standard value can bedetermined based on the expression level of the above-mentioned markergene in the control. For example, in certain embodiments, thepermissible range is taken as ±2S.D. based on the standard value. Oncethe standard value is determined, the testing method may be performed bymeasuring only the expression level in a biological sample from asubject and comparing the value with the determined standard value forthe control.

Expression levels of marker genes include transcription of the markergenes to mRNA, and translation into proteins. Therefore, one method oftesting for a colon cancer-related disease is performed based on acomparison of the intensity of expression of mRNA corresponding to themarker genes, or the expression level of proteins encoded by the markergenes.

The measurement of the expression levels of marker genes in the testingfor a colon cancer-related disease can be carried out according tovarious gene analysis methods. Specifically, one can use, for example, ahybridization technique using nucleic acids that hybridize to thesegenes as probes, or a gene amplification technique using DNA thathybridize to the marker genes as primers.

The probes or primers used for the testing can be designed based on thenucleotide sequences of the marker genes. The identification numbers forthe nucleotide sequences of the respective marker genes are describerherein.

Further, it is to be understood that genes of higher animals generallyaccompany polymorphism in a high frequency. There are also manymolecules that produce isoforms comprising mutually different amino acidsequences during the splicing process. Any gene associated with a coloncancer-related disease that has an activity similar to that of a markergene is included in the marker genes, even if it has nucleotide sequencedifferences due to polymorphism or being an isoform.

It is also to be understood that the marker genes can include homologsof other species in addition to humans. Thus, unless otherwisespecified, the expression “marker gene” refers to a homolog of themarker gene unique to the species or a foreign marker gene which hasbeen introduced into an individual.

Also, it is to be understood that a “homolog of a marker gene” refers toa gene derived from a species other than a human, which can hybridize tothe human marker gene as a probe under stringent conditions. Suchstringent conditions are known to one skilled in the art who can selectan appropriate condition to produce an equal stringency experimentallyor empirically.

A polynucleotide comprising the nucleotide sequence of a marker gene ora nucleotide sequence that is complementary to the complementary strandof the nucleotide sequence of a marker gene and has at least 15nucleotides, can be used as a primer or probe. Thus, a “complementarystrand” means one strand of a double stranded DNA with respect to theother strand and which is composed of A:T (U for RNA) and G:C basepairs.

In addition, “complementary” means not only those that are completelycomplementary to a region of at least 15 continuous nucleotides, butalso those that have a nucleotide sequence homology of at least 40% incertain instances, 50% in certain instances, 60% in certain instances,70% in certain instances, at least 80%, 90%, and 95% or higher. Thedegree of homology between nucleotide sequences can be determined by analgorithm, BLAST, etc.

Such polynucleotides are useful as a probe to detect a marker gene, oras a primer to amplify a marker gene. When used as a primer, thepolynucleotide comprises usually 15 bp to 100 bp, and in certainembodiments 15 bp to 35 bp of nucleotides. When used as a probe, a DNAcomprises the whole nucleotide sequence of the marker gene (or thecomplementary strand thereof), or a partial sequence thereof that has atleast 15 bp nucleotides. When used as a primer, the 3′ region must becomplementary to the marker gene, while the 5′ region can be linked to arestriction enzyme-recognition sequence or a tag.

“Polynucleotides” may be either DNA or RNA. These polynucleotides may beeither synthetic or naturally-occurring. Also, DNA used as a probe forhybridization is usually labeled. Those skilled in the art readilyunderstand such labeling methods. Herein, the term “oligonucleotide”means a polynucleotide with a relatively low degree of polymerization.Oligonucleotides are included in polynucleotides.

Tests for a colon cancer-related disease using hybridization techniquescan be performed using, for example, Northern hybridization, dot blothybridization, or the DNA microarray technique. Furthermore, geneamplification techniques, such as the RT-PCR method may be used. Byusing the PCR amplification monitoring method during the geneamplification step in RT-PCR, one can achieve a more quantitativeanalysis of the expression of a marker gene.

In the PCR gene amplification monitoring method, the detection target(DNA or reverse transcript of RNA) is hybridized to probes that arelabeled with a fluorescent dye and a quencher which absorbs thefluorescence. When the PCR proceeds and Taq polymerase degrades theprobe with its 5′-3′ exonuclease activity, the fluorescent dye and thequencher draw away from each other and the fluorescence is detected. Thefluorescence is detected in real time. By simultaneously measuring astandard sample in which the copy number of a target is known, it ispossible to determine the copy number of the target in the subjectsample with the cycle number where PCR amplification is linear. Also,one skilled in the art recognizes that the PCR amplification monitoringmethod can be carried out using any suitable method.

The method of testing for a colon cancer-related disease can be alsocarried out by detecting a protein encoded by a marker gene.Hereinafter, a protein encoded by a marker gene is described as a“marker protein.” For such test methods, for example, the Westernblotting method, the immunoprecipitation method, and the ELISA methodmay be employed using an antibody that binds to each marker protein.

Antibodies used in the detection that bind to the marker protein may beproduced by any suitable technique. Also, in order to detect a markerprotein, such an antibody may be appropriately labeled. Alternatively,instead of labeling the antibody, a substance that specifically binds tothe antibody, for example, protein A or protein G, may be labeled todetect the marker protein indirectly. More specifically, such adetection method can include the ELISA method.

A protein or a partial peptide thereof used as an antigen may beobtained, for example, by inserting a marker gene or a portion thereofinto an expression vector, introducing the construct into an appropriatehost cell to produce a transformant, culturing the transformant toexpress the recombinant protein, and purifying the expressed recombinantprotein from the culture or the culture supernatant. Alternatively, theamino acid sequence encoded by a gene or an oligopeptide comprising aportion of the amino acid sequence encoded by a full-length cDNA arechemically synthesized to be used as an immunogen.

Furthermore, a test for a colon cancer-related disease can be performedusing as an index not only the expression level of a marker gene butalso the activity of a marker protein in a biological sample. Activityof a marker protein means the biological activity intrinsic to theprotein. Various methods can be used for measuring the activity of eachprotein.

Even if a patient is not diagnosed as being affected with a coloncancer-related disease in a routine test in spite of symptoms suggestingthese diseases, whether or not such a patient is suffering from a coloncancer-related disease can be easily determined by performing a testaccording to the methods described herein.

More specifically, in certain embodiments, when the marker gene is oneof the genes described herein, an increase or decrease in the expressionlevel of the marker gene in a patient whose symptoms suggest at least asusceptibility to a colon cancer-related disease indicates that thesymptoms are primarily caused by a colon cancer-related disease.

In addition, the tests are useful to determine whether a coloncancer-related disease is improving in a patient. In other words, themethods described herein can be used to judge the therapeutic effect ofa treatment for a colon cancer-related disease. Furthermore, when themarker gene is one of the genes described herein, an increase ordecrease in the expression level of the marker gene in a patient, whohas been diagnosed as being affected by a colon cancer-related disease,implies that the disease has progressed more.

The severity and/or susceptibility to a colon cancer-related disease mayalso be determined based on the difference in expression levels. Forexample, when the marker gene is one of the genes described herein, thedegree of increase in the expression level of the marker gene iscorrelated with the presence and/or severity of a colon cancer-relateddisease.

Animal Models

In another aspect, there is provided herein animal models for a coloncancer-related disease where the expression level of one or more markergenes or a gene functionally equivalent to the marker gene has beenelevated in the animal model. A “functionally equivalent gene” as usedherein generally is a gene that encodes a protein having an activitysimilar to a known activity of a protein encoded by the marker gene. Arepresentative example of a functionally equivalent gene includes acounterpart of a marker gene of a subject animal, which is intrinsic tothe animal.

The animal model for a colon cancer-related disease is useful fordetecting physiological changes due to a colon cancer-related disease.In certain embodiments, the animal model is useful to reveal additionalfunctions of marker genes and to evaluate drugs whose targets are themarker genes.

In one embodiment, an animal model for a colon cancer-related diseasecan be created by controlling the expression level of a counterpart geneor administering a counterpart gene. The method can include creating ananimal model for a colon cancer-related disease by controlling theexpression level of a gene selected from the group of genes describedherein. In another embodiment, the method can include creating an animalmodel for a colon cancer-related disease by administering the proteinencoded by a gene described herein, or administering an antibody againstthe protein. It is to be also understood, that in certain otherembodiments, the marker can be over-expressed such that the marker canthen be measured using appropriate methods.

In another embodiment, an animal model for a colon cancer-relateddisease can be created by introducing a gene selected from such groupsof genes, or by administering a protein encoded by such a gene.

In another embodiment, a colon cancer-related disease can be induced bysuppressing the expression of a gene selected from such groups of genesor the activity of a protein encoded by such a gene. An antisensenucleic acid, a ribozyme, or an RNAi can be used to suppress theexpression. The activity of a protein can be controlled effectively byadministering a substance that inhibits the activity, such as anantibody.

The animal model is useful to elucidate the mechanism underlying a coloncancer-related disease and also to test the safety of compounds obtainedby screening. For example, when an animal model develops the symptoms ofcolon cancer-related disease, or when a measured value involved in acertain a colon cancer-related disease alters in the animal, a screeningsystem can be constructed to explore compounds having activity toalleviate the disease.

As used herein, the expression “an increase in the expression level”refers to any one of the following: where a marker gene introduced as aforeign gene is expressed artificially; where the transcription of amarker gene intrinsic to the subject animal and the translation thereofinto the protein are enhanced; or where the hydrolysis of the protein,which is the translation product, is suppressed. As used herein, theexpression “a decrease in the expression level” refers to either thestate in which the transcription of a marker gene of the subject animaland the translation thereof into the protein are inhibited, or the statein which the hydrolysis of the protein, which is the translationproduct, is enhanced. The expression level of a gene can be determined,for example, by a difference in signal intensity on a DNA chip.Furthermore, the activity of the translation product—the protein—can bedetermined by comparing with that in the normal state.

It is also within the contemplated scope that the animal model caninclude transgenic animals, including, for example animals where amarker gene has been introduced and expressed artificially; marker geneknockout animals; and knock-in animals in which another gene has beensubstituted for a marker gene. A transgenic animal, into which anantisense nucleic acid of a marker gene, a ribozyme, a polynucleotidehaving an RNAi effect, or a DNA functioning as a decoy nucleic acid orsuch has been introduced, can be used as the transgenic animal. Suchtransgenic animals also include, for example, animals in which theactivity of a marker protein has been enhanced or suppressed byintroducing a mutation(s) into the coding region of the gene, or theamino acid sequence has been modified to become resistant or susceptibleto hydrolysis. Mutations in an amino acid sequence includesubstitutions, deletions, insertions, and additions.

In addition, the expression itself of a marker gene can be controlled byintroducing a mutation(s) into the transcriptional regulatory region ofthe gene. Those skilled in the art understand such amino acidsubstitutions. Also, the number of amino acids that are mutated is notparticularly restricted, as long as the activity is maintained Normally,it is within 50 amino acids, in certain non-limiting embodiments, within30 amino acids, within 10 amino acids, or within 3 amino acids. The siteof mutation may be any site, as long as the activity is maintained.

In yet another aspect, there is provided herein screening methods forcandidate compounds for therapeutic agents to treat a coloncancer-related disease. One or more marker genes are selected from thegroup of genes described herein. A therapeutic agent for a coloncancer-related disease can be obtained by selecting a compound capableof increasing or decreasing the expression level of the marker gene(s).

It is to be understood that the expression “a compound that increasesthe expression level of a gene” refers to a compound that promotes anyone of the steps of gene transcription, gene translation, or expressionof a protein activity. On the other hand, the expression “a compoundthat decreases the expression level of a gene”, as used herein, refersto a compound that inhibits any one of these steps.

In particular aspects, the method of screening for a therapeutic agentfor a colon cancer-related disease can be carried out either in vivo orin vitro. This screening method can be performed, for example, by (1)administering a candidate compound to an animal subject; (2) measuringthe expression level of a marker gene(s) in a biological sample from theanimal subject; or (3) selecting a compound that increases or decreasesthe expression level of a marker gene(s) as compared to that in acontrol with which the candidate compound has not been contacted.

In still another aspect, there is provided herein a method to assess theefficacy of a candidate compound for a pharmaceutical agent on theexpression level of a marker gene(s) by contacting an animal subjectwith the candidate compound and monitoring the effect of the compound onthe expression level of the marker gene(s) in a biological samplederived from the animal subject. The variation in the expression levelof the marker gene(s) in a biological sample derived from the animalsubject can be monitored using the same technique as used in the testingmethod described above. Furthermore, based on the evaluation, acandidate compound for a pharmaceutical agent can be selected byscreening.

All patents, patent applications and references cited herein areincorporated in their entirety by reference. While the invention hasbeen described and exemplified in sufficient detail for those skilled inthis art to make and use it, various alternatives, modifications andimprovements should be apparent without departing from the spirit andscope of the invention. One skilled in the art readily appreciates thatthe present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those inherenttherein.

The methods and reagents described herein are representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Modifications therein andother uses will occur to those skilled in the art. These modificationsare encompassed within the spirit of the invention and are defined bythe scope of the claims. It will also be readily apparent to a personskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

It should be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modifications and variations of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

What is claimed is:
 1. A method of diagnosing whether a subject has apoor survival prognosis colon adenocarcinoma, comprising: measuring thelevel of at least one miR-181b gene product in a test sample from thesubject wherein said subject has colon adenocarcinoma, wherein anincrease in at least the level of the miR-181b gene product in the testsample, relative to the level of a corresponding miR-181b gene productin a control sample, is indicative of the subject having poor survivalprognosis colon adenocarcinoma.
 2. A method of testing for poor survivalprognosis colon adenocarcinoma, which comprises: (1) determining anexpression level of at least one marker in a sample from a test subjecthaving colon adenocarcinoma; the at least one marker including at leastone miR-181b gene product; (2) comparing the expression level determinedin step (1) with a control expression level of the marker in a samplefrom a healthy subject; and (3) judging the subject to have a poorsurvival prognosis colon adenocarcinoma when the result of thecomparison in step (2) indicates that: the expression level of the atleast one marker in the test subject is higher than that in the control.3. The testing method of claim 2, wherein the sample comprises one ormore of tissue, blood, plasma, serum, urine, and feces.
 4. The testingmethod of claim 2, wherein all method steps are performed in vitro.
 5. Amethod of diagnosing whether a subject has poor survival prognosis colonadenocarcinoma, comprising: (1) reverse transcribing RNA from a testsample obtained from the subject to provide a set of targetoligodeoxynucleotides wherein said subject has colon adenocarcinoma; (2)hybridizing the target oligodeoxynucleotides to a microarray comprisingmiRNA-181b specific probe oligonucleotides to provide a hybridizationprofile for the test sample; and (3) comparing the test samplehybridization profile to a hybridization profile generated from acontrol sample, wherein an increase in the signal of the miR-181b isindicative of the subject having poor survival prognosis colonadenocarcinoma.
 6. The method of claim 1, wherein a level of expressionof miR-181b gene product is assessed by detecting the presence of atranscribed polynucleotide or portion thereof, wherein the transcribedpolynucleotide comprises a coding region of miR-181b gene product. 7.The method of claim 1, wherein the sample is a colon cancer-associatedbody fluid or tissue.
 8. The method of claim 1, wherein the samplecomprises cells obtained from the patient.
 9. The method of claim 1,wherein the at least one miR-181b gene product includes isolatedvariants or biologically-active fragments thereof.
 10. A method of claim1, which further comprises measuring the level of at least oneadditional miR gene product in the test sample, wherein the miR isselected from the group consisting of: miR-21; let-7g; miR-16b;miR-106a; miR-103-2; miR-203; miR-29a; and miR-10a.
 11. A method ofclaim 1, which further comprises measuring the level of at least two ormore additional miR gene products in the test sample, wherein the miRsare selected from the group consisting of: miR-21; let-7g; miR-16b;miR-106a; miR-103-2; miR-203; miR-29a; and miR-10a.
 12. A method ofclaim 1, which further comprises measuring the level of at least threeor more additional miR gene products in the test sample, wherein themiRs are selected from the group consisting of: miR-21; let-7g; miR-16b;miR-106a; miR-103-2; miR-203; miR-29a; and miR-10a.
 13. A method ofclaim 2, which further comprises measuring the level of at least oneadditional miR gene product in the test sample, wherein the miR isselected from the group consisting of: miR-21; let-7g; miR-16b;miR-106a; miR-103-2; miR-203; miR-29a; and miR-10a.
 14. A method ofclaim 2, which further comprises measuring the level of at least two ormore additional miR gene products in the test sample, wherein the miRsare selected from the group consisting of: miR-21; let-7g; miR-16b;miR-106a; miR-103-2; miR-203; miR-29a; and miR-10a.
 15. A method ofclaim 2, which further comprises measuring the level of at least threeor more additional miR gene products in the test sample, wherein themiRs are selected from the group consisting of: miR-21; let-7g; miR-16b;miR-106a; miR-103-2; miR-203; miR-29a; and miR-10a.
 16. A method ofclaim 5, which further comprises measuring the level of at least oneadditional miR gene product in the test sample, wherein the miR isselected from the group consisting of: miR-21; let-7g; miR-16b;miR-106a; miR-103-2; miR-203; miR-29a; and miR-10a.
 17. A method ofclaim 5, which further comprises measuring the level of at least two ormore additional miR gene products in the test sample, wherein the miRsare selected from the group consisting of: miR-21; let-7g; miR-16b;miR-106a; miR-103-2; miR-203; miR-29a; and miR-10a.
 18. A method ofclaim 5, which further comprises measuring the level of at least threeor more additional miR gene products in the test sample, wherein themiRs are selected from the group consisting of: miR-21; let-7g; miR-16b;miR-106a; miR-103-2; miR-203; miR-29a; and miR-10a.